new interesting findings

http://link.springer.com/article/10.1007/s10722-015-0254-2

Size matters: evolution of large drug-secreting resin glands
in elite pharmaceutical strains of Cannabis sativa (marijuana)
Ernest Small . Steve G. U. Naraine
Genet Resour Crop Evol

----------------------------------------------
http://link.springer.com/article/10.1007/s10722-015-0253-3

Expansion of female sex organs in response to prolonged
virginity in Cannabis sativa (marijuana)
Ernest Small . Steve G. U. Naraine
Genet Resour Crop Evol

-------------------------------------------------



http://www.sciencedirect.com/science/article/pii/S0031942215001181
tetrahydrocannabinolic acid-synthase in Cannabis sativa L. and its relationship with chemical phenotype
Chiara Onofri a, Etienne P.M. de Meijer b, Giuseppe Mandolino a,
a Consiglio per la ricerca in agricoltura e l’analisi dell’economia agraria, Centro di Ricerca per le Colture Industriali, via di Corticella 133, 40128 Bologna, Italy
b GW Pharmaceuticals PLC, Ground Floor South Wing, Kingsgate House, Newbury Road, Andover SP10 4DU, United Kingdom

Received 2 October 2014, Revised 21 March 2015, Available online 9 April 2015

Abstract
Sequence variants of THCA- and CBDA-synthases were isolated from different Cannabis sativa L. strains expressing various wild-type and mutant chemical phenotypes (chemotypes). Expressed and complete sequences were obtained from mature inflorescences. Each strain was shown to have a different specificity and/or ability to convert the precursor CBGA into CBDA and/or THCA type products. The comparison of the expressed sequences led to the identification of different mutations, all of them due to SNPs. These SNPs were found to relate to the cannabinoid composition of the inflorescence at maturity and are therefore proposed to have a functional significance. The amount of variation was found to be higher within the CBDAS sequence family than in the THCAS family, suggesting a more recent evolution of THCA-forming enzymes from the CBDAS group. We therefore consider CBDAS as the ancestral type of these synthases.
-----------
Does this mean THC synthase evolved or mutated from CBD synthase? Maybe man helped select the few that were THC synthase however they came to be, and spread them worldwide? I would not be surprised......
Man selects for THC and against CBD, in the case of Ganja, he selects for high resin contents regardless of Cannabinoid for Hashish, for hemp until recently there was little selections for Cannabinoids until after Cannabis became illegal less then 100 years ago, and very recently for CBD for mediacal varieties.
-SamS

---------------


http://onlinelibrary.wiley.com/doi/10.1111/bph.12944/full

Are cannabidiol and Δ9-tetrahydrocannabivarin negative modulators of the endocannabinoid system? A systematic review

John M McPartland1, Marnie Duncan, Vincenzo Di Marzo and Roger G Pertwee
Article first published online: 8 JAN 2015

© 2014 The British Pharmacological Society
Issue
British Journal of Pharmacology
Volume 172, Issue 3, pages 737–753, February 2015

Abstract
Based upon evidence that the therapeutic properties of Cannabis preparations are not solely dependent upon the presence of Δ9-tetrahydrocannabinol (THC), pharmacological studies have been recently carried out with other plant cannabinoids (phytocannabinoids), particularly cannabidiol (CBD) and Δ9-tetrahydrocannabivarin (THCV). Results from some of these studies have fostered the view that CBD and THCV modulate the effects of THC via direct blockade of cannabinoid CB1 receptors, thus behaving like first-generation CB1 receptor inverse agonists, such as rimonabant. Here, we review in vitro and ex vivo mechanistic studies of CBD and THCV, and synthesize data from these studies in a meta-analysis. Synthesized data regarding mechanisms are then used to interpret results from recent pre-clinical animal studies and clinical trials. The evidence indicates that CBD and THCV are not rimonabant-like in their action and thus appear very unlikely to produce unwanted CNS effects. They exhibit markedly disparate pharmacological profiles particularly at CB1 receptors: CBD is a very low-affinity CB1 ligand that can nevertheless affect CB1 receptor activity in vivo in an indirect manner, while THCV is a high-affinity CB1 receptor ligand and potent antagonist in vitro and yet only occasionally produces effects in vivo resulting from CB1 receptor antagonism. THCV has also high affinity for CB2 receptors and signals as a partial agonist, differing from both CBD and rimonabant. These cannabinoids illustrate how in vitro mechanistic studies do not always predict in vivo pharmacology and underlie the necessity of testing compounds in vivo before drawing any conclusion on their functional activity at a given target.

---------------------------------------------------------

http://www.sciencedirect.com/science/article/pii/S003194221200475X

Phytochemistry
Volume 87, March 2013, Pages 51–59


Analysis of cannabinoids in laser-microdissected trichomes of medicinal Cannabis sativa using LCMS and cryogenic NMR

Nizar Happyanaa, b, Sara Agnoletc, Remco Muntendamd, Annie Van Dame, Bernd Schneiderc, Oliver Kaysera, ,
a Department of Technical Biochemistry, Technical University of Dortmund, Technische Biochemie, Emil-Figge-Strasse 66, D-44227 Dortmund, Germany
b Department of Chemistry, Bandung Institute of Technology, Jl. Ganesha 10, Bandung 40132, Indonesia
c Research Group Biosynthesis/NMR, Max Planck Institute for Chemical Ecology, Hans-Knöll-Straße 8, 07745 Jena, Germany
d Department of Pharmaceutical Biology, University of Groningen, Antonius Deusinglaan 1, 9713 AV, Groningen, The Netherlands
e Mass Spectrometry Core Facility, Center for Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV, Groningen, The Netherlands



Abstract
Trichomes, especially the capitate-stalked glandular hairs, are well known as the main sites of cannabinoid and essential oil production of Cannabis sativa. In this study the distribution and density of various types of Cannabis sativa L. trichomes, have been investigated by scanning electron microscopy (SEM). Furthermore, glandular trichomes were isolated over the flowering period (8 weeks) by laser microdissection (LMD) and the cannabinoid profile analyzed by LCMS. Cannabinoids were detected in extracts of 25–143 collected cells of capitate-sessile and capitate stalked trichomes and separately in the gland (head) and the stem of the latter. Δ9-Tetrahydrocannabinolic acid [THCA (1)], cannabidiolic acid [CBDA (2)], and cannabigerolic acid [CBGA (3)] were identified as most-abundant compounds in all analyzed samples while their decarboxylated derivatives, Δ9-tetrahydrocannabinol [THC (4)], cannabidiol [CBD (5)], and cannabigerol [CBG (6)], co-detected in all samples, were present at significantly lower levels. Cannabichromene [CBC (8)] along with cannabinol (CBN (9)) were identified as minor compounds only in the samples of intact capitate-stalked trichomes and their heads harvested from 8-week old plants. Cryogenic nuclear magnetic resonance spectroscopy (NMR) was used to confirm the occurrence of major cannabinoids, THCA (1) and CBDA (2), in capitate-stalked and capitate-sessile trichomes. Cryogenic NMR enabled the additional identification of cannabichromenic acid [CBCA (7)] in the dissected trichomes, which was not possible by LCMS as standard was not available. The hereby documented detection of metabolites in the stems of capitate-stalked trichomes indicates a complex biosynthesis and localization over the trichome cells forming the glandular secretion unit.

--------------------------------------

http://www.sciencedirect.com/science/article/pii/S0926669013000526

Industrial Crops and Products
Volume 46, April 2013, Pages 269–273

Ethephon application stimulats cannabinoids and plastidic terpenoids production in Cannabis sativa at flowering stage
Hakimeh Mansouri, , Fatemeh Salari, Zahra Asrar
Received 27 November 2012, Accepted 4 January 2013, Available online 1 March 2013


Abstract
We studied the effect of ethephon on levels of the major cannabinoids (tetrahydrocannabinol and cannabidiol) and chlorophyll, carotenoids and α-tocopherol in Cannabis sativa at productive stage. Results revealed that ethephon increased THC content of leaf in male and female plants and of male flowers. However, ethephon unable to enhancing THC content in female flowers. Treatment with etheohon increased CBD content in male and female leaf and female flowers. The treatment of male flowers with low ethephon concentration caused an increase, and those treated with high ethephon concentration resulted in a decrease in CBD content. The lowest level of ethephon (1 μM) enhanced chlorophyll a, b and total chlorophyll in male and female plants. Both sexes treated with ethephon showed an increase in carotenoids content, but 1 μM ethephon had the stronger effect in this regards. Male and female plants had a higher content of α-tocopherol when treated with ethephon. These results showed ethephon is a suitable treatment for increasing cannabinoids and α-tocopherol in productive stage of cannabis and there was not a relation between primary and secondary terpenoids.

Highlights
► Ethephon treatment has considerable effects on increasing of cannabinoids in male and female cannabis. ► There is no correlation between cannabinoids and other plastidial terpenoids. ► Using of ethephon treatment in flowering stage do not adverse effect on plant growth.


http://www.ncbi.nlm.nih.gov/pubmed/26000707

J Nat Prod. 2015 May 22. [Epub ahead of print]
Isolation and Pharmacological Evaluation of Minor Cannabinoids from High-Potency Cannabis sativa.
Radwan MM1, ElSohly MA1, El-Alfy AT1, Ahmed SA1, Slade D1, Husni AS1, Manly SP1, Wilson L1, Seale S1, Cutler SJ1, Ross SA1.


Abstract
Seven new naturally occurring hydroxylated cannabinoids (1-7), along with the known cannabiripsol (8), have been isolated from the aerial parts of high-potency Cannabis sativa. The structures of the new compounds were determined by 1D and 2D NMR spectroscopic analysis, GC-MS, and HRESIMS as 8α-hydroxy-Δ9-tetrahydrocannabinol (1), 8β-hydroxy-Δ9-tetrahydrocannabinol (2), 10α-hydroxy-Δ8-tetrahydrocannabinol (3), 10β-hydroxy-Δ8-tetrahydrocannabinol (4), 10α-hydroxy-Δ9,11-hexahydrocannabinol (5), 9β,10β-epoxyhexahydrocannabinol (6), and 11-acetoxy-Δ9-tetrahydrocannabinolic acid A (7). The binding affinity of isolated compounds 1-8, Δ9-tetrahydrocannabinol, and Δ8-tetrahydrocannabinol toward CB1 and CB2 receptors as well as their behavioral effects in a mouse tetrad assay were studied. The results indicated that compound 3, with the highest affinity to the CB1 receptors, exerted the most potent cannabimimetic-like actions in the tetrad assay, while compound 4 showed partial cannabimimetic actions. Compound 2, on the other hand, displayed a dose-dependent hypolocomotive effect only.

Good stuff. That second paper is right up my alley. Here is a link to the free full-text:

Are cannabidiol and Δ9-tetrahydrocannabivarin negative modulators of the endocannabinoid system? A systematic review


This paper reflects the growing maturity of pharmacological studies, away from simplistic models based solely on binding affinities at a couple of receptors, and towards a multivariate analysis that takes into consideration the "wider canvas" of receptors and the crosstalk between them, allosteric modulation, enzymes, transporters, transcription factors, etc. I also like the trend towards polypharmacology that makes promiscuity a virtue.

It also touches on some stuff I am particularly interested in- adenosine receptors and the heteromers they form with other GPCRs (especially metabotropic glutamate receptors). I think the negative effects I get from too much CBD (bradycardia and vertigo mainly) are due to the functional adenosine receptor agonism of CBD, as coffee (and other xanthine rich foods) helps lessen them.

The four boxes listing the landmark studies are nice too, as is the exhaustive references section.

Thanks, I added it to my post.
-SamS

mofeta said:

Good stuff. That second paper is right up my alley. Here is a link to the free full-text:

Are cannabidiol and Δ9-tetrahydrocannabivarin negative modulators of the endocannabinoid system? A systematic review


This paper reflects the growing maturity of pharmacological studies, away from simplistic models based solely on binding affinities at a couple of receptors, and towards a multivariate analysis that takes into consideration the "wider canvas" of receptors and the crosstalk between them, allosteric modulation, enzymes, transporters, transcription factors, etc. I also like the trend towards polypharmacology that makes promiscuity a virtue.

It also touches on some stuff I am particularly interested in- adenosine receptors and the heteromers they form with other GPCRs (especially metabotropic glutamate receptors). I think the negative effects I get from too much CBD (bradycardia and vertigo mainly) are due to the functional adenosine receptor agonism of CBD, as coffee (and other xanthine rich foods) helps lessen them.

The four boxes listing the landmark studies are nice too, as is the exhaustive references section.

Click to expand...

THE FIRST TWO ARE NOT CANNABIS SPECIFIC BUT INTERESTING TO ME.



http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0128808

Large-Scale Evolutionary Analysis of Genes and Supergene Clusters from Terpenoid Modular Pathways Provides Insights into Metabolic Diversification in Flowering Plants
Johannes A. Hofberger, Aldana M. Ramirez, Erik van den Bergh, Xinguang Zhu, Harro J. Bouwmeester, Robert C. Schuurink, M. Eric Schranz

Abstract
An important component of plant evolution is the plethora of pathways producing more than 200,000 biochemically diverse specialized metabolites with pharmacological, nutritional and ecological significance. To unravel dynamics underlying metabolic diversification, it is critical to determine lineage-specific gene family expansion in a phylogenomics framework. However, robust functional annotation is often only available for core enzymes catalyzing committed reaction steps within few model systems. In a genome informatics approach, we extracted information from early-draft gene-space assemblies and non-redundant transcriptomes to identify protein families involved in isoprenoid biosynthesis. Isoprenoids comprise terpenoids with various roles in plant-environment interaction, such as pollinator attraction or pathogen defense. Combining lines of evidence provided by synteny, sequence homology and Hidden-Markov-Modelling, we screened 17 genomes including 12 major crops and found evidence for 1,904 proteins associated with terpenoid biosynthesis. Our terpenoid genes set contains evidence for 840 core terpene-synthases and 338 triterpene-specific synthases. We further identified 190 prenyltransferases, 39 isopentenyl-diphosphate isomerases as well as 278 and 219 proteins involved in mevalonate and methylerithrol pathways, respectively. Assessing the impact of gene and genome duplication to lineage-specific terpenoid pathway expansion, we illustrated key events underlying terpenoid metabolic diversification within 250 million years of flowering plant radiation. By quantifying Angiosperm-wide versatility and phylogenetic relationships of pleiotropic gene families in terpenoid modular pathways, our analysis offers significant insight into evolutionary dynamics underlying diversification of plant secondary metabolism. Furthermore, our data provide a blueprint for future efforts to identify and more rapidly clone terpenoid biosynthetic genes from any plant species.



http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0129598

Transcriptomic Analysis Reveals Possible Influences of ABA on Secondary Metabolism of Pigments, Flavonoids and Antioxidants in Tomato Fruit during Ripening
Wangshu Mou, Dongdong Li, Zisheng Luo, Linchun Mao, Tiejin Ying

Abstract
Abscisic acid (ABA) has been proven to be involved in the regulation of climacteric fruit ripening, but a comprehensive investigation of its influence on ripening related processes is still lacking. By applying the next generation sequencing technology, we conducted a comparative analysis of the effects of exogenous ABA and NDGA (Nordihydroguaiaretic acid, an inhibitor of ABA biosynthesis) on tomato fruit ripening. The high throughput sequencing results showed that out of the 25728 genes expressed across all three samples, 10388 were identified as significantly differently expressed genes. Exogenous ABA was found to enhance the transcription of genes involved in pigments metabolism, including carotenoids biosynthesis and chlorophyll degradation, whereas NDGA treatment inhibited these processes. The results also revealed the crucial role of ABA in flavonoids synthesis and regulation of antioxidant system. Intriguingly, we also found that an inhibition of endogenous ABA significantly enhanced the transcriptional abundance of genes involved in photosynthesis. Our results highlighted the significance of ABA in regulating tomato ripening, which provided insight into the regulatory mechanism of fruit maturation and senescence process.




http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0110638

Diversity Analysis in Cannabis sativa Based on Large-Scale Development of Expressed Sequence Tag-Derived Simple Sequence Repeat Markers
Chunsheng Gao , Pengfei Xin , Chaohua Cheng, Qing Tang, Ping Chen, Changbiao Wang, Gonggu Zang, Lining Zhao

Abstract
Cannabis sativa L. is an important economic plant for the production of food, fiber, oils, and intoxicants. However, lack of sufficient simple sequence repeat (SSR) markers has limited the development of cannabis genetic research. Here, large-scale development of expressed sequence tag simple sequence repeat (EST-SSR) markers was performed to obtain more informative genetic markers, and to assess genetic diversity in cannabis (Cannabis sativa L.). Based on the cannabis transcriptome, 4,577 SSRs were identified from 3,624 ESTs. From there, a total of 3,442 complementary primer pairs were designed as SSR markers. Among these markers, trinucleotide repeat motifs (50.99%) were the most abundant, followed by hexanucleotide (25.13%), dinucleotide (16.34%), tetranucloetide (3.8%), and pentanucleotide (3.74%) repeat motifs, respectively. The AAG/CTT trinucleotide repeat (17.96%) was the most abundant motif detected in the SSRs. One hundred and seventeen EST-SSR markers were randomly selected to evaluate primer quality in 24 cannabis varieties. Among these 117 markers, 108 (92.31%) were successfully amplified and 87 (74.36%) were polymorphic. Forty-five polymorphic primer pairs were selected to evaluate genetic diversity and relatedness among the 115 cannabis genotypes. The results showed that 115 varieties could be divided into 4 groups primarily based on geography: Northern China, Europe, Central China, and Southern China. Moreover, the coefficient of similarity when comparing cannabis from Northern China with the European group cannabis was higher than that when comparing with cannabis from the other two groups, owing to a similar climate. This study outlines the first large-scale development of SSR markers for cannabis. These data may serve as a foundation for the development of genetic linkage, quantitative trait loci mapping, and marker-assisted breeding of cannabis.



http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0085118

Molecular Cytogenetic Characterization of the Dioecious Cannabis sativa with an XY Chromosome Sex Determination System
Mikhail G. Divashuk, Oleg S. Alexandrov, Olga V. Razumova, Ilya V. Kirov, Gennady I. Karlov

Abstract
Hemp (Cannabis sativa L.) was karyotyped using by DAPI/C-banding staining to provide chromosome measurements, and by fluorescence in situ hybridization with probes for 45 rDNA (pTa71), 5S rDNA (pCT4.2), a subtelomeric repeat (CS-1) and the Arabidopsis telomere probes. The karyotype has 18 autosomes plus a sex chromosome pair (XX in female and XY in male plants). The autosomes are difficult to distinguish morphologically, but three pairs could be distinguished using the probes. The Y chromosome is larger than the autosomes, and carries a fully heterochromatic DAPI positive arm and CS-1 repeats only on the less intensely DAPI-stained, euchromatic arm. The X is the largest chromosome of all, and carries CS-1 subtelomeric repeats on both arms. The meiotic configuration of the sex bivalent locates a pseudoautosomal region of the Y chromosome at the end of the euchromatic CS-1-carrying arm. Our molecular cytogenetic study of the C. sativa sex chromosomes is a starting point for helping to make C. sativa a promising model to study sex chromosome evolution.



http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0099641

Understanding Cultivar-Specificity and Soil Determinants of the Cannabis Microbiome
Max E. Winston , Jarrad Hampton-Marcell, Iratxe Zarraonaindia, Sarah M. Owens, Corrie S. Moreau, Jack A. Gilbert, Josh Hartsel, Suzanne J. Kennedy, S. M. Gibbons

Abstract
Understanding microbial partnerships with the medicinally and economically important crop Cannabis has the potential to affect agricultural practice by improving plant fitness and production yield. Furthermore, Cannabis presents an interesting model to explore plant-microbiome interactions as it produces numerous secondary metabolic compounds. Here we present the first description of the endorhiza-, rhizosphere-, and bulk soil-associated microbiome of five distinct Cannabis cultivars. Bacterial communities of the endorhiza showed significant cultivar-specificity. When controlling cultivar and soil type the microbial community structure was significantly different between plant cultivars, soil types, and between the endorhiza, rhizosphere and soil. The influence of soil type, plant cultivar and sample type differentiation on the microbial community structure provides support for a previously published two-tier selection model, whereby community composition across sample types is determined mainly by soil type, while community structure within endorhiza samples is determined mainly by host cultivar.



http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0120016

Impacts of Surface Water Diversions for Marijuana Cultivation on Aquatic Habitat in Four Northwestern California Watersheds
Scott Bauer , Jennifer Olson , Adam Cockrill, Michael van Hattem, Linda Miller, Margaret Tauzer, Gordon Leppig

Abstract
Marijuana (Cannabis sativa L.) cultivation has proliferated in northwestern California since at least the mid-1990s. The environmental impacts associated with marijuana cultivation appear substantial, yet have been difficult to quantify, in part because cultivation is clandestine and often occurs on private property. To evaluate the impacts of water diversions at a watershed scale, we interpreted high-resolution aerial imagery to estimate the number of marijuana plants being cultivated in four watersheds in northwestern California, USA. Low-altitude aircraft flights and search warrants executed with law enforcement at cultivation sites in the region helped to validate assumptions used in aerial imagery interpretation. We estimated the water demand of marijuana irrigation and the potential effects water diversions could have on stream flow in the study watersheds. Our results indicate that water demand for marijuana cultivation has the potential to divert substantial portions of streamflow in the study watersheds, with an estimated flow reduction of up to 23% of the annual seven-day low flow in the least impacted of the study watersheds. Estimates from the other study watersheds indicate that water demand for marijuana cultivation exceeds streamflow during the low-flow period. In the most impacted study watersheds, diminished streamflow is likely to have lethal or sub-lethal effects on state-and federally-listed salmon and steelhead trout and to cause further decline of sensitive amphibian species.

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http://www.ncbi.nlm.nih.gov/pubmed/26048837

Talanta
Volume 140, 1 August 2015, Pages 150–165

1H NMR and HPLC/DAD for Cannabis sativa L. chemotype distinction, extract profiling and specification
Wieland Peschel, , Matteo Poli

Highlights
•
1H NMR (DMSO-d6) key signals allow distinction of THC, CBD and CBG type cannabis.
•
We detect neutral/acidic cannabinoids and phenolics co-compounds via HPLC/DAD.
•
Non-heated extract profiles suggest consideration of cannabinoid acids.
•
Cell viability reduction correlated with the total cannabinoid content but not THC.
•
We suggest markers for drug distinction and pharmaceutical specification.
Abstract
The medicinal use of different chemovars and extracts of Cannabis sativa L. requires standardization beyond ∆9-tetrahydrocannabinol (THC) with complementing methods. We investigated the suitability of 1H NMR key signals for distinction of four chemotypes measured in deuterated dimethylsulfoxide together with two new validated HPLC/DAD methods used for identification and extract profiling based on the main pattern of cannabinoids and other phenolics alongside the assayed content of THC, cannabidiol (CBD), cannabigerol (CBG) their acidic counterparts (THCA, CBDA, CBGA), cannabinol (CBN) and cannflavin A and B. Effects on cell viability (MTT assay, HeLa) were tested. The dominant cannabinoid pairs allowed chemotype recognition via assignment of selective proton signals and via HPLC even in cannabinoid-low extracts from the THC, CBD and CBG type. Substantial concentrations of cannabinoid acids in non-heated extracts suggest their consideration for total values in chemotype distinction and specifications of herbal drugs and extracts. Cannflavin A/B are extracted and detected together with cannabinoids but always subordinated, while other phenolics can be accumulated via fractionation and detected in a wide fingerprint but may equally serve as qualitative marker only. Cell viability reduction in HeLa was more determined by the total cannabinoid content than by the specific cannabinoid profile. Therefore the analysis and labeling of total cannabinoids together with the content of THC and 2–4 lead cannabinoids are considered essential. The suitability of analytical methods and the range of compound groups summarized in group and ratio markers are discussed regarding plant classification and pharmaceutical specification.



http://www.ncbi.nlm.nih.gov/pubmed/23890639

Forensic Science International
Volume 231, Issues 1–3, 10 September 2013, Pages 208–212


Analysis of THCA synthase gene expression in cannabis: A preliminary study by real-time quantitative PCR
Fidelia Cascinia, Stella Passerottib, Ilaria Boschia

Abstract
In this paper we describe analyses performed by Real-Time Reverse-Transcriptase Polymerase Chain Reaction (real-time RT-PCR) on RNA of 12 samples, carried out for forensic purposes to investigate a correlation between tetrahydrocannabinol (THC) concentration in Cannabis and the tetrahydrocannabinol acid synthase (THCAS) gene expression.

Samples were obtained from an experimental cultivation of declared potency Cannabis variety seeds and from seizures. The Rubisco gene and the 26S ribosomal RNA gene were used as internal control genes for their constant expression and stability.

As results we found minor gene expression in samples from leaves of young plants.

Further, grouping results for cannabis samples with similar characteristics, we have found an increased relative expression in samples with the highest percentage of THC coming from seized sample and adult plants.





http://neuron.mefst.hr/docs/CMJ/issues/2003/44/3/12808725.pdf

Encyclopedia of Forensic Sciences (Second Edition)
2013, Pages 382–386

Biology/DNA/Botany

Cannabis DNA Typing Methods
H. Miller Coyle

Abstract
This article focuses on Cannabis, primarily, the development of biomarkers to classify and uniquely identify marijuana. DNA methods such as short tandem repeat, amplified fragment length polymorphism, and single-nucleotide polymorphism are discussed with an emphasis on forensic data-basing applications and the benefits and limitations of each method. It also provides an overview of marijuana regarding the history of cultivation in the United States, associative evidence at crime scenes and current law enforcement concerns in the US, and microscopic and chemical methods for classification, geo-sourcing and high-throughput screening.




http://www.researchgate.net/profile/Ornelio_Rosati/publications

Studies in Natural Products Chemistry
Volume 45, 2015, Pages 17–57

Chapter 2 – Cannabis and Bioactive Cannabinoids
Federica Messina, Ornelio Rosati, Massimo Curini, M. Carla Marcotulli

Abstract
The therapeutic use of Cannabis dates back to ancient times and this plant has been used for centuries as remedy for a large number of diseases. Today it is well known that biological activity of Cannabis is related to the endocannabinoid system (ECS), a complex signaling network that comprises classical cannabinoid receptors (CB1 and CB2), arachidonic acid-derived ligands, and enzymes degrading the endocannabinoids anandamide and 2-arachidonoyl glycerol, namely fatty acid amide hydrolase and monoacylglycerol lipase. The modulation of the ECS activity turned out to be a therapeutic promise in a wide range of diseases. A problem to the development of Cannabis and cannabinoid medications is the psychoactive property of natural or synthetic agonists, mediated by CB1 receptor. This review deals with the literature analysis of the important biological activities of Cannabis and the efforts aimed to the discovery of natural and nonnatural selective cannabinoids.




http://www.ncbi.nlm.nih.gov/pubmed/24528581

Forensic Science International: Genetics
Volume 9, March 2014, Pages 61–65


Characterization of 15 STR cannabis loci: Nomenclature proposal and SNPSTR haplotypes
Laura Valverdea, Christian Lischkaa, Stefanie Scheipera, Johanna Nedelea, Rachel Challisa, Marian M. de Pancorbob, Heidi Pfeiffera, Stephan Köhnemanna,

Abstract
The standardization of methods for individualizing Cannabis sativa plants could offer new possibilities in the investigation of its illegal trade. Here we present the first nomenclature proposal for 15 cannabis STRs, which allows an initial standardization for performing comparisons between laboratories and generating genotype databases. Several alleles of the 15 STR loci have been sequenced. This has revealed that not all the STR loci are equally suitable for the individualization purposes. Moreover, several nucleotide variations have been detected both inside the repeat structure and/or in the flanking region. All the different SNPSTR haplotypes are presented and compared with the previous sequence raw data of the 15 STR loci. The SNPSTR data could considerably increase the informative value of the STRs, which could be very useful in complex cases.





http://www.sciencedirect.com/science/book/9780124186798

The Effects of Drug Abuse on the Human Nervous System
2014, Pages 387–422


Chapter Thirteen – Effects of Cannabis and Cannabinoids in the Human Nervous System
Harold Kalant

Abstract
The endocannabinoid (EC) system, consisting of ECs, their synthesizing and degrading enzymes, specific transmembrane EC transporters and receptors, is located in both excitatory and inhibitory synapses of all the classical neurotransmitter types throughout the central and peripheral nervous systems, where it acts as a retrograde signaling mechanism to inhibit further release of transmitter. This form of synaptic plasticity is a major component of both rapid short-term and sustained long-term adaptive responses that underlie such processes as homeostasis, learning, memory, and extinction. The functional effects on any given pathway can be either inhibitory or excitatory, depending on whether excitatory (e.g., glutamatergic) or inhibitory (e.g., GABAergic) modulation normally predominates in that pathway. However, the dose-effect curves of EC activity are in many instances biphasic, because sustained strong activity leads to EC receptor desensitization and down-regulation, resulting in progressive loss or even reversal of the effect. Therefore the effects of cannabis and exogenous cannabinoids, of both plant and synthetic origin, are in many cases different from, or even opposite to, those of the EC system.

The functional effects of the EC system and of exogenous cannabinoids are compared with respect to neuronal growth and maturation, neuroprotection against toxic and traumatic damage, sensory pathways, nausea and vomiting, appetite and food intake, the sleep/wake cycle, affective responses and mood states, motor control, seizure activity and cognitive functions. Effects in laboratory animals are compared to those in humans, including both actual and potential therapeutic effects and adverse effects. The therapeutic effects in most instances correspond to the low-dose actions of the EC system, whereas the adverse effects generally correspond to the high-dose range. The exogenous cannabinoids are less selective in their actions than the EC system because they act on a much wider range of EC receptors throughout the nervous system. It is concluded that for most potential therapeutic applications the future will lie with the development of highly selective site-specific agents that act on individual components of the EC system, rather than on the whole system.





http://www.sciencedirect.com/science/article/pii/S0379073815001449

Forensic Science International
Volume 251, June 2015, Pages 95–106


Evaluation of elemental profiling methods, including laser-induced breakdown spectroscopy (LIBS), for the differentiation of Cannabis plant material grown in different nutrient solutions
Moteaa M. El-Deftara, , , James Robertsona, , Simon Fosterb, , Chris Lennardc,

Highlights
•
Application of LIBS, ICP-MS, LA-ICP-MS and μXRF for the analysis of Cannabis plant.
•
Discrimination of Cannabis is evaluated based on their elemental composition data.
•
It is possible to correctly associate Cannabis with a particular nutrient.
•
ICP-MS, LA-ICP-MS and LIBS are suitable methods for the analysis of Cannabis plant.
Abstract
Laser-induced breakdown spectroscopy (LIBS) is an emerging atomic emission based solid sampling technique that has many potential forensic applications. In this study, the analytical performance of LIBS, as well as that of inductively coupled plasma mass spectrometry (ICP-MS), laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) and X-ray microfluorescence (μXRF), was evaluated for the ability to conduct elemental analyses on Cannabis plant material, with a specific investigation of the possible links between hydroponic nutrients and elemental profiles from associated plant material. No such study has been previously published in the literature. Good correlation among the four techniques was observed when the concentrations or peak areas of the elements of interest were monitored. For Cannabis samples collected at the same growth time, the elemental profiles could be related to the use of particular commercial nutrients. In addition, the study demonstrated that ICP-MS, LA-ICP-MS and LIBS are suitable techniques for the comparison of Cannabis samples from different sources, with high discriminating powers being achieved. On the other hand, μXRF method was not suitable for the discrimination of Cannabis samples originating from different growth nutrients.






http://www.sciencedirect.com/science/article/pii/S0021967314016173

Journal of Chromatography A
Volume 1370, 28 November 2014, Pages 200–215


Multidimensional analysis of cannabis volatile constituents: Identification of 5,5-dimethyl-1-vinylbicyclo[2.1.1]hexane as a volatile marker of hashish, the resin of Cannabis sativa L.
Marie Marchinia, Céline Charvozb, Laurence Dujourdyb, Nicolas Baldovinia, Jean-Jacques Filippia, ,

Highlights
•
Analysis of cannabis herb and hashish volatile constituents by HS-SPME-GC × GC–MS.
•
Identification of a new volatile marker of hashish.
•
Photolytic rearrangement of β-myrcene into hashishene.
•
Formation of photo-oxidation products during hashish manufacture.
Abstract
The volatile constituents of drug samples derived from Cannabis sativa L. were investigated by means of headspace solid phase microextraction (HS-SPME) and gas chromatography techniques (GC–MS, GC × GC–MS). Samples of cannabis herb and hashish showed clear differences in their volatile chemical profiles, mostly resulting from photo-oxidation processes occurring during the transformation of fresh cannabis herb into hashish. Most unexpectedly, we could demonstrate hashish samples as containing remarkable amounts of a rare and unusual monoterpene – 5,5-dimethyl-1-vinylbicyclo[2.1.1]hexane – among the volatile compounds detected in their headspaces. We gave evidence for the formation of this compound from the light induced rearrangement of β-myrcene during the manufacture of hashish. In view of its high abundance among volatile constituents of cannabis resin and its scarce occurrence in other natural volatile extracts, we propose to rename this specific monoterpene hashishene.





http://www.sciencedirect.com/science/article/pii/S2090536X14000525

Egyptian Journal of Forensic Sciences
Available online 11 November 2014


Open Access
GCMS analysis of Cannabis sativa L. from four different areas of Pakistan
Muhammad Tayyab, , Durre Shahwar

Abstract
Cannabis is most frequently used drug of abuse not only in Pakistan but also in the whole world. Its use is mounting drastically every year. GCMS allows analysis of Cannabis sativa which shows divergence of the constituents of this plant. Prevalence of this plant can be identified through knowledge of its constituents. In this way we can obstruct the production if we know the region in which it is produced. GCMS is a useful technique for the comparison of constituents of this drug of abuse which will assist the investigator concerning the origin of plant. Comparison also aids in the understanding and acquaintance of similarities of different samples of cannabinoids.



http://link.springer.com/article/10.1007/s11738-011-0874-x

Acta Physiologiae Plantarum
March 2012, Volume 34, Issue 2, pp 743-750
Date: 02 Dec 2011
In vitro germplasm conservation of high Δ9 -tetrahydrocannabinol yielding elite clones of Cannabis sativa L. under slow growth conditions
Hemant Lata, Suman Chandra, Zlatko Mehmedic, Ikhlas A. Khan, Mahmoud A. ElSohly

Abstract
Germplasm conservation of a high Δ9-tetrahydrocannabinol yielding variety of Cannabis sativa L. was attempted using synthetic seed technology and media supplemented with osmotic agents. Explants of nodal segments containing single axillary bud were excised from in vitro proliferated shoot cultures and encapsulated in high-density sodium alginate (230 mM) hardened by 50 mM CaCl2. The ‘encapsulated’ (synthetic seeds) and ‘non-encapsulated’ nodal segments were stored at 5, 15 and 25°C for 8, 16 and 24 weeks and monitored for the re-growth and survival frequency under the tissue culture conditions (16-h photoperiod, 25°C) on Murashige and Skoog (MS) medium supplemented with thidiazuron (TDZ 0.5 μM). ‘Encapsulated’ nodal segments could be stored at low temperature 15°C up to 24 weeks with maximum re-growth ability and survival frequency of 60%. Similar to ‘encapsulated’ cultures, the highest re-growth in ‘non-encapsulated’ cultures was observed in the explants kept at 15°C without osmotic agents. Furthermore, the effect of osmotic agents mannitol and sorbitol (2 and 4% w/v, added individually and in combination to the media at culture room conditions i.e. 25°C) on non-encapsulated shoot cultures was also evaluated. A considerable decrease in re-growth and survival was observed in the cultures treated with osmotic agents. Among the cultures treated with different concentrations of osmotic agents, the highest rate of re-growth and survival was observed at the lowest concentration of 2% sorbitol and 2% mannitol individually added to the media. Well-developed plantlets regenerated from ‘encapsulated’ nodal segments were successfully acclimatized inside the growing room with 90% survival frequency. Gas chromatography-flame ionization detection (GC-FID) was used to compare the chemical profile and the concentration of the different cannabinoids (cannabidiol, cannabichromene, cannabigerol, cannabinol, Δ9-tetrahydrocannabinol and tetrahydrocannabivarin) of the plants grown from ‘encapsulated’ nodal segments to that of the donor plant. The data showed similar cannabinoid profile and insignificant differences in the cannabinoids content between the two types of plants. This study is of high significance since the encapsulation technology would allow the prolonged storage (thus reducing the cost of labor) of high-yielding C. sativa germplasm selected for the isolation of THC, a high-value bulk active pharmaceutic.



http://link.springer.com/article/10.1007/s00705-011-1168-8

Arch Virol DOI 10.1007/s00705-011-1168-8
Complete sequence of a cryptic virus from hemp (Cannabis sativa)
Angelika Ziegler • Jaroslav Matousˇek •
Gerhard Steger • Jorg Schubert
Received: 9 August 2011 / Accepted: 29 October 2011
Springer-Verlag 2011
Abstract
Hemp (Cannabis sativa) was found to be a
useful propagation host for hop latent virus, a carlavirus.
However, when virus preparations were analysed by elec-
tron microscopy, along with the expected filamentous
particles, spherical particles with a diameter of around
34 nm were found. RNA from virus preparations was
purified, and cDNA was prepared and cloned. Sequence
information was used to search databases, and the greatest
similarity was found with Primula malacoides virus 1, a
putative new member of the genus Partitivirus. The full
sequences of RNA 1 and RNA 2 of this new hemp cryptic
virus were obtained.

Nice findes! I liked especially the "Diversity Analysis in Cannabis sativa...".
What would actually be nice is some sort of real life connection. For example, how we might use the findings from the ABA paper when growing cannabis.

EDIT: Just realised that this publication has a major flaw: The 'European' hemp selection is heavily based on Fibrimon and 'Soviet' (Russia, Ukraine) varieties including the northern ecotype. This geographically places the 'European' hemp close to the northern Chinese varieties and their similarity has nothing to do with a similar climate. Though the latter is true per se, the test employed should not or at least only to a minor degree and more by chance than anything else reflect climate (i.e. phenotype) but only the degree of relationship. Basically, their test is a parenthood test. You're not all of a sudden related with Ali or Chen just because they live in a similar climate. And that's what I meant in the other thread (recent findings blabla) with 'forced publications' from Asia. In order to try not being a racist I didn't say illiterate, uneducated, and devoid of logical thinking outside of the box but basically, it's just that with many of the Asian PhD students. A good part is not really studied the way we're used to. On one hand, they're 40 years old mothers whose husband is rich enough to pay them a PhD and on another, they can not think different than their professor but treat him like an all knowing deity (who often doesn't care what exactly his stuff publishes or he simply doesn't understand it because it's too modern). Whatever, be twice as careful with publications not from Europe or North America as you should be when you read the latter .

Only Ornamental said:

What would actually be nice is some sort of real life connection. For example, how we might use the findings from the ABA paper when growing cannabis. .

Click to expand...

This would really be a new interesting finding!

shag

That paper about ABA is rather useless for us as tomato fruits have not too much in common with MJ.
Several other things in there, as for example the shift in the metabolic pathway of phenylalanine is already known. Unfortunately, I haven't come to a useful conclusion how to use that except for faster lignification (might be good to produce bonsai plants??).
The helpful stuff about ABA might be compiled from other publications but I'm too lazy to do that here (mostly because I have no ABA and don't really believe in its benefits yet... maybe it could be used as an antidote for GA side effects or to induce early flowering whilst keeping stretch in check?). But that's worth its own thread.

PS Hi Shaggy,
Nice seeing you around!! Howzit hangin'?

Only Ornamental said:

PS Hi Shaggy,
Nice seeing you around!! Howzit hangin'?

Click to expand...

Hey OO

I aint been round much of late due to personal issues.
But it is good to be back, if only for a moment.
It is always good to hear your point of view OO.
It usually differs from the normal rants you commonly find here!

Not pointing fingers or casting judgment, just sayin'

Peace to all
see ya'll soon.

Shag

Can someone give me the Cliff notes on this paper or break it down real simple for a lazy simpleton like me?

I saw the big sexy calyxes from no sexy time and then it got boring.

Or wait until it comes out on DVD....
-SamS

yesum said:

Can someone give me the Cliff notes on this paper or break it down real simple for a lazy simpleton like me?

I saw the big sexy calyxes from no sexy time and then it got boring.

Click to expand...

Sam, sometimes I do understand why you don't fancy my proposition that much .

Thanks for the links, it's a pity that most aren't accessible for free though. Knowledge is getting expensive nowadays! lol.

Vibes

Some are available at other sites for free, like ResearchGate or PLOS but you may need to join the site to get full access.
Any paper I really want I find a way, I ask the authors for a copy, that often works. Or from an academic friend, but yes they are expensive if you bought them all, I don't hardly buy any, I find a way. If anyone sees any of them online for free let me know, so we can share the link info.

-SamS

one more new one:
I find interesting as CBD has been reported to not bind to the CB1 yet it does modulate THC, this is how.
-SamS

http://www.ncbi.nlm.nih.gov/pubmed/26218440

Br J Pharmacol. 2015 Jul 27.
Cannabidiol is a negative allosteric modulator of the type 1 cannabinoid receptor.

BACKGROUND AND PURPOSE:
Cannabidiol has been reported to act as an antagonist of cannabinoid agonists at type 1 cannabinoid receptors (CB1 ). We hypothesized that cannabidiol can inhibit cannabinoid agonist activity through negative allosteric modulation of CB1 .

http://dx.doi.org/10.4172/2329-6836.1000181

Open access full text!!

Natural Products Chemistry & Research
Cannabinoids and Terpenes as Chemotaxonomic Markers in Cannabis
Elzinga S, Fischedick J, Podkolinski R, and Raber J

Abstract
In this paper, we present principal component analysis (PCA) results from a dataset containing 494 cannabis
flower samples and 170 concentrate samples analyzed for 31 compounds. A continuum of chemical composition
amongst cannabis strains was found instead of distinct chemotypes. Our data shows that some strains are much
more reproducible in chemical composition than others. Strains labeled as indica were compared with those labeled
as sativa and no evidence was found that these two cultivars are distinctly different chemotypes. PCA of “OG” and
“Kush” type strains found that “OG” strains have relatively higher levels of α-terpineol, fenchol, limonene, camphene, terpinolene and linalool where “Kush” samples are characterized mainly by the compounds trans-ocimene, guaiol, β-eudesmol, myrcene and α-pinene. The composition of concentrates and flowers were compared as well. Although the absolute concentration of compounds in concentrates is much higher, the relative composition of compounds between flowers and concentrates is similar.

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http://link.springer.com/article/10.1007/s12229-015-9157-3


The Botanical Review
August 2015
Date: 19 Aug 2015
Evolution and Classification of Cannabis sativa (Marijuana, Hemp) in Relation to Human Utilization
Ernest Small

Abstract
Cannabis sativa has been employed for thousands of years, primarily as a source of a stem fiber (both the plant and the fiber termed “hemp”) and a resinous intoxicant (the plant and its drug preparations commonly termed “marijuana”). Studies of relationships among various groups of domesticated forms of the species and wild-growing plants have led to conflicting evolutionary interpretations and different classifications, including splitting C. sativa into several alleged species. This review examines the evolving ways Cannabis has been used from ancient times to the present, and how human selection has altered the morphology, chemistry, distribution and ecology of domesticated forms by comparison with related wild plants. Special attention is given to classification, since this has been extremely contentious, and is a key to understanding, exploiting and controlling the plant. Differences that have been used to recognize cultivated groups within Cannabis are the results of disruptive selection for characteristics selected by humans. Wild-growing plants, insofar as has been determined, are either escapes from domesticated forms or the results of thousands of years of widespread genetic exchange with domesticated plants, making it impossible to determine if unaltered primeval or ancestral populations still exist. The conflicting approaches to classifying and naming plants with such interacting domesticated and wild forms are examined. It is recommended that Cannabis sativa be recognized as a single species, within which there is a narcotic subspecies with both domesticated and ruderal varieties, and similarly a non-narcotic subspecies with both domesticated and ruderal varieties. An alternative approach consistent with the international code of nomenclature for cultivated plants is proposed, recognizing six groups: two composed of essentially non-narcotic fiber and oilseed cultivars as well as an additional group composed of their hybrids; and two composed of narcotic strains as well as an additional group composed of their hybrids.

I like that last one on the OG, thanks!

One more

One more

http://www.plosone.org/article/fetc....1371/journal.pone.0133292&representation=PDF

The Genetic Structure of Marijuana and Hemp

Jason Sawler, Jake M. Stout, Kyle M. Gardner, Darryl Hudson, John Vidmar, Laura Butler, Jonathan E. Page, Sean Myles PLOS x
Published: August 26, 2015DOI: 10.1371/journal.pone.0133292

Despite its cultivation as a source of food, fibre and medicine, and its global status as the most used illicit drug, the genus Cannabis has an inconclusive taxonomic organization and evolutionary history. Drug types of Cannabis (marijuana), which contain high amounts of the psychoactive cannabinoid Δ9-tetrahydrocannabinol (THC), are used for medical purposes and as a recreational drug. Hemp types are grown for the production of seed and fibre, and contain low amounts of THC. Two species or gene pools (C. sativa and C. indica) are widely used in describing the pedigree or appearance of cultivated Cannabis plants. Using 14,031 single-nucleotide polymorphisms (SNPs) genotyped in 81 marijuana and 43 hemp samples, we show that marijuana and hemp are significantly differentiated at a genome-wide level, demonstrating that the distinction between these populations is not limited to genes underlying THC production. We find a moderate correlation between the genetic structure of marijuana strains and their reported C. sativa and C. indica ancestry and show that marijuana strain names often do not reflect a meaningful genetic identity. We also provide evidence that hemp is genetically more similar to C. indica type marijuana than to C. sativa strains.

http://www.genomebiology.com/2011/12/10/R102

The draft genome and transcriptome of Cannabis sativa
Genome Biol. 2011 Oct 20;12(10):R102. doi: 10.1186/gb-2011-12-10-r102.
Harm van Bakel, Jake M Stout, Atina G Cote, Carling M Tallon, Andrew G Sharpe, Timothy R Hughes and Jonathan E Page

Here are a few more, new and old. A lot are Molecular Biology, not really my forte. If anyone has links to the full papers post them and I will add the links.
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Purification and characterization of cannabidiolic-acid synthase from Cannabis sativa L. Biochemical analysis of a novel enzyme that catalyzes the oxidocyclization of cannabigerolic acid to cannabidiolic acid

Futoshi Taura, S Morimoto, Yukihiro Shoyama
Faculty of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812, Japan.
Journal of Biological Chemistry . 08/1996; 271(29):17411-6. DOI:*10.1074/jbc.271.29.17411

ABSTRACT We identified a unique enzyme that catalyzes the oxidocyclization of cannabigerolic acid to cannabidiolic acid (CBDA) in Cannabis sativa L. (CBDA strain). The enzyme, named CBDA synthase, was purified to apparent homogeneity by a four-step procedure: ammonium sulfate precipitation followed by chromatography on DEAE-cellulose, phenyl-Sepharose CL-4B, and hydroxylapatite. The active enzyme consists of a single polypeptide with a molecular mass of 74 kDa and a pI of 6.1. The NH2-terminal amino acid sequence of CBDA synthase is similar to that of Delta1-tetrahydrocannabinolic-acid synthase. CBDA synthase does not require coenzymes, molecular oxygen, hydrogen peroxide, and metal ion cofactors for the oxidocyclization reaction. These results indicate that CBDA synthase is neither an oxygenase nor a peroxidase and that the enzymatic cyclization does not proceed via oxygenated intermediates. CBDA synthase catalyzes the formation of CBDA from cannabinerolic acid as well as cannabigerolic acid, although the kcat for the former (0.03 s-1) is lower than that for the latter (0.19 s-1). Therefore, we conclude that CBDA is predominantly biosynthesized from cannabigerolic acid rather than cannabinerolic acid.

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Tetrahydrocannabinolic acid synthase, the enzyme controlling marijuana psychoactivity is secreted into the storage cavity of the glandular trichomes.

Supaart Sirikantaramas, Futoshi Taura, Yumi Tanaka, Yu Ishikawa, Satoshi Morimoto, Yukihiro Shoyama
Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582 Japan.
Plant and Cell Physiology . 10/2005; 46(9):1578-82. DOI:*10.1093/pcp/pci166

ABSTRACT Tetrahydrocannabinolic acid (THCA) synthase is the enzyme responsible for the production of tetrahydrocannabinol (THC), the psychoactive component of marijuana (Cannabis sativa L.). We suggest herein that THCA is biosynthesized in the storage cavity of the glandular trichomes based on the following observations. (i) The exclusive expression of THCA synthase was confirmed in the secretory cells of glandular trichomes by reverse transcription-PCR (RT-PCR) analysis. (ii) THCA synthase activity was detected in the storage cavity content. (iii) Transgenic tobacco expressing THCA synthase fused to green fluorescent protein showed fluorescence in the trichome head corresponding to the storage cavity. These results also showed that secretory cells of the glandular trichomes secrete not only metabolites but also biosynthetic enzyme.


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Identification and Characterization of Cannabinoids That Induce Cell Death through Mitochondrial Permeability Transition in Cannabis Leaf Cells
Satoshi Morimoto 1 , Yumi Tanaka, Kaori Sasaki, Hiroyuki Tanaka, Tomohide Fukamizu, Yoshinari Shoyama, Yukihiro Shoyama and Futoshi Taura

Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan

Cannabinoids are secondary metabolites stored in capitate-sessile glands on leaves of Cannabis sativa. We discovered that cell death is induced in the leaf tissues exposed to cannabinoid resin secreted from the glands, and identified cannabichromenic acid (CBCA) and Δ1-tetrahydrocannabinolic acid (THCA) as unique cell death mediators from the resin. These cannabinoids effectively induced cell death in the leaf cells or suspension-cultured cells of C. sativa, whereas pretreatment with the mitochondrial permeability transition (MPT) inhibitor cyclosporin A suppressed this cell death response. Examinations using isolated mitochondria demonstrated that CBCA and THCA mediate opening of MPT pores without requiring Ca2+ and other cytosolic factors, resulting in high amplitude mitochondrial swelling, release of mitochondrial proteins (cytochrome c and nuclease), and irreversible loss of mitochondrial membrane potential. Therefore, CBCA and THCA are considered to cause serious damage to mitochondria through MPT. The mitochondrial damage was also confirmed by a marked decrease of ATP level in cannabinoid-treated suspension cells. These features are in good accord with those of necrotic cell death, whereas DNA degradation was also observed in cannabinoid-mediated cell death. However, the DNA degradation was catalyzed by nuclease(s) released from mitochondria during MPT, indicating that this reaction was not induced via a caspase-dependent apoptotic pathway. Furthermore, the inhibition of the DNA degradation only slightly blocked the cell death induced by cannabinoids. Based on these results, we conclude that CBCA and THCA have the ability to induce necrotic cell death via mitochondrial dysfunction in the leaf cells of C. sativa.

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Cannabinoids Production by Hairy Root Cultures of Cannabis sativa L.
Sayed Farag, Oliver Kayser*
American Journal of Plant Sciences, 2015, 6, 1874-1884
Technische Biochemie, Fachbereich Bio- und Chemieingenieurwesen, Technische Universität Dortmund, Dortmund, Germany
Abstract:
Tetrahydrocannabinol (THC) derivatives are used clinically as analgesic, anti-inflammatory, appetite stimulant, anti-emetic and anti-tumor cannabinoids. THC and its related compounds are at present obtained by extraction from intact Cannabis plants or chemical synthesis, but plant cell cultures may be an alternative source of production. In the present study, hairy root cultures of C. sativa (Cannabaceae) were induced by incubation of aseptically grown callus culture with solid B5 medium supplemented with 4 mg/l naphthaleneacetic acid in darkness at 25 ̊C. Hairy root growth profiles in shake flask, increased periodically during 35 days of growth cycle. The cannabinoid contents produced in minor levels and remained below 2.0 μg/g dry weight. The contents of can-
nabinoid were analyzed by liquid chromatography and confirmed by mass spectrometry.

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Production of D9-tetrahydrocannabinolic acid from cannabigerolic acid by whole cells of Pichia (Komagataella) pastoris expressing D9-tetrahydrocannabinolic acid synthase from Cannabis sativa L.
Bastian Zirpel . Felix Stehle . Oliver Kayser
Biotechnol Lett (2015) 37:1869–1875
DOI 10.1007/s10529-015-1853-x
Abstract
Objective The D9-tetrahydrocannabinolic acid synthase (THCAS) from Cannabis sativa was expressed
intracellularly in different organisms to investigate the potential of a biotechnological production of D9-
tetrahydrocannabinolic acid (THCA) using whole cells. Results Functional expression of THCAS was ob-
tained in Saccharomyces cerevisiae and Pichia (Ko- magataella) pastoris using a signal peptide from the
vacuolar protease, proteinase A. No functional expression was achieved in Escherichia coli. The highest
volumetric activities obtained were 98 pkat ml-1 (intracellular) and 44 pkat ml-1 (extracellular) after
192 h of cultivation at 15 C using P. pastoris cells. Low solubility of CBGA prevents the THCAS appli-
cation in aqueous cell-free systems, thus whole cells were used for a bioconversion of cannabigerolic acid
(CBGA) to THCA. Finally, 1 mM (0.36 g THCA l-1) THCA could be produced by 10.5 gCDW l-1 before
enzyme activity was lost.
Conclusion
Whole cells of P. pastoris offer the capability of synthesizing pharmaceutical THCA production

----------------------------------------------------------------------

Molecular analysis of genetic fidelity in Cannabis sativa L. plants grown from synthetic (encapsulated) seeds following in vitro storage
Biotechnology Letters
December 2011, Volume 33, Issue 12, pp 2503-2508
Hemant Lata, Suman Chandra , Natascha Techen, Ikhlas A. Khan, Mahmoud A. ElSohly

Abstract
The increasing utilization of synthetic (encapsulated) seeds for germplasm conservation and propagation necessitates the assessment of genetic stability of conserved propagules following their plantlet conversion. We have assessed the genetic stability of synthetic seeds of Cannabis sativa L. during in vitro multiplication and storage for 6 months at different growth conditions using inter simple sequence repeat (ISSR) DNA fingerprinting. Molecular analysis of randomly selected plants from each batch was conducted using 14 ISSR markers. Of the 14 primers tested, nine produced 40 distinct and reproducible bands. All the ISSR profiles from in vitro stored plants were monomorphic and comparable to the mother plant which confirms the genetic stability among the clones. GC analysis of six major cannabinoids [Δ9-tetrahydrocannabinol, tetrahydrocannabivarin, cannabidiol, cannabichromene, cannabigerol and cannabinol] showed homogeneity in the re-grown clones and the mother plant with insignificant differences in cannabinoids content, thereby confirming the stability of plants derived from synthetic seeds following 6 months storage.

----------------------------------------

Recent Advances in Cannabis sativa Research: Biosynthetic Studies and Its Potential in Biotechnology.

Supaart Sirikantaramas, Futoshi Taura, Satoshi Morimoto, Yukihiro Shoyama
Graduate School of Pharmaceutical Sciences, Kyushu University, Higashi-ku, Fukuoka, Japan.
Current pharmaceutical biotechnology. 09/2007; 8(4):237-43. DOI:*10.2174/138920107781387456
Source: PubMed
ABSTRACT:
Cannabinoids, consisting of alkylresorcinol and monoterpene groups, are the unique secondary metabolites that are found only in Cannabis sativa. Tetrahydrocannabinol (THC), cannabidiol (CBD) and cannabichromene (CBC) are well known cannabinoids and their pharmacological properties have been extensively studied. Recently, biosynthetic pathways of these cannabinoids have been successfully established. Several biosynthetic enzymes including geranylpyrophosphatelivetola te geranyltransferase, tetrahydrocannabinolic acid (THCA) synthase, cannabidiolic acid (CBDA) synthase and cannabichromenic acid (CBCA) synthase have been purified from young rapidly expanding leaves of C. sativa. In addition, molecular cloning, characterization and localization of THCA synthase have been recently reported. THCA and cannabigerolic acid (CBGA), its substrate, were shown to be apoptosis-inducing agents that might play a role in plant defense. Transgenic tobacco hairy roots expressing THCA synthase can produce THCA upon feeding of CBGA. These results open the way for biotechnological production of cannabinoids in the future.

------------------------------------------------------------

Production of Δ1-tetrahydrocannabinolic acid by the biosynthetic enzyme secreted from transgenic Pichia pastoris

Futoshi Taura, Emi Dono, Supaart Sirikantaramas, Kohji Yoshimura, Yukihiro Shoyama,
Satoshi Morimoto
Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan.
Biochemical and Biophysical Research Communications (Impact Factor: 2.28). 10/2007; 361(3):675-80. DOI:*10.1016/j.bbrc.2007.07.079

ABSTRACT Delta(1)-Tetrahydrocannabinolic acid (THCA) synthase is the enzyme that catalyzes the oxidative cyclization of cannabigerolic acid into THCA, the acidic precursor of Delta(1)-tetrahydrocannabinol. We developed a novel expression system for THCA synthase using a methylotrophic yeast Pichia pastoris as a host. Under optimized conditions, the transgenic P. pastoris secreted approximately 1.32nkat/l of THCA synthase activity, and the culture medium, from which the cells were removed, effectively synthesized THCA from cannabigerolic acid with a approximately 98% conversion rate. The secreted THCA synthase was readily purified to homogeneity. Interestingly, endoglycosidase treatment afforded a deglycosylated THCA synthase with more catalytic activity than that of the glycosylated form. The non-glycosylated THCA synthase should be suitable for structure-function studies because it displayed much more activity than the previously reported native enzyme from Cannabis sativa as well as the recombinant enzyme from insect cell cultures.
----------------------------------------

Hello everybody,

Nature published an outlook on cannabis
and I thought this could make a good sunday read :

http://www.nature.com/nature/journal/v525/n7570_supp/index.html?WT.mc_id=FBK_1509_NAT_CANNABIS

This contains 10 outlook and 4 related articles. If this is in the wrong section, please move/remove since I'm not entirely sure that this qualifies as a new and interesting finding .... Anyhow enjoy the read.

Cheers