Evaluation of microsatellites of Catha edulis (qat; Celastraceae) identified using pyrosequencing

The use of Next Generation Sequencing (NGS) techniques to identify microsatellite markers has replaced more time intensive methods such as molecular cloning. The main advantage of NGS over traditional methods of identifying microsatellite markers is the generation of many more sequences with less effort. It is possible to design primers from unenriched DNA, thereby further reducing the workload and also allowing the use of SSRs that are difficult to enrich (e.g., TA/AT and TAA/ATT). We present microsatellite primer pairs that may be used for phylogeographic analysis as well as to infer the geographical origin of traded material of Catha edulis, which contains two amphetamines that are controlled substances in many counties. We used data from two partial 454 pyrosequencing runs that generated about 2000 sequences containing microsatellites (3% of all sequences) as well as flanking regions sufficient for primer design. Using 23 samples of C. edulis we identified 27 single-copy markers that were broadly amplified across the sampled individuals; 18 showed polymorphism information content (PIC) higher than 0.5. The genetic structure in wild individuals is concordant with their geographic origins; wild samples from northern Kenya are more closely related to Ethiopian samples than are other wild samples from Kenya. The geographic differences in allele frequencies indicate that microsatellite analysis can be used to determine the geographic source of cultivated and wild collected material.     

(-)-N-formylnorephedrine from Catha edulis

The alkaloidal fraction of Catha edulis yielded upon repeated chromatography (-)-N-formylnorephedrine whose ¹HNMR and ¹³CNMR spectra suggested the presence of cisoid (major) and transoid forms (minor). The identity of the isolated compound was established by comparison with the major product obtained by formylating (-)-norephedrine; the minor product was found to be (-)-N,O-diformylnorephedrine.     

Phylogeny of the Celastreae (Celastraceae) and the relationships of Catha edulis (qat) inferred from morphological characters and nuclear and plastid genes

The phylogeny of Celastraceae tribe Celastreae, which includes about 350 species of trees and shrubs in 15 genera, was inferred in a simultaneous analysis of morphological characters together with nuclear (ITS and 26S rDNA) and plastid (matK, trnL-F) genes. A strong correlation was found between the geography of the species sampled and their inferred relationships. Species of Maytenus and Gymnosporia from different regions were resolved as polyphyletic groups. Maytenus was resolved in three lineages (New World, African, and Austral-Pacific), while Gymnosporia was resolved in two lineages (New World and Old World). Putterlickia was resolved as nested within the Old World Gymnosporia. Catha edulis (qat, khat) was resolved as sister to the clade of Allocassine, Cassine, Lauridia, and Maurocenia. Gymnosporia cassinoides, which is reportedly chewed as a stimulant in the Canary Islands, was resolved as a derived member of Gymnosporia and is more closely related to Lydenburgia and Putterlickia than it is to Catha. Therefore, all eight of these genera are candidates for containing cathinone- and/or cathine-related alkaloids.     

Mycotoxin-producing potential of fungi isolated from red kidney beans

The predominant fungi present in samples of reject and retail red kidney beans were Aspergillus glaucus, Penicillium spp. and Alternaria spp. Together with A. ochraceus, A. flavus, Fusarium spp., and Trichoderma, these isolates from the reject beans were screened for numerous mycotoxins by TLC. The most consistently produced mycotoxins were penicillic acid (from A. ochraceus and Penicillium spp.) and Alternaria toxins (tenuazonic acid and alternariol). A. glaucus strains were tested for cytotoxicity in three tissue culture cell lines with positive results.     

The psychostimulant drug khat (Catha edulis): A mini-review

Khat (Catha edulis Forsk) is a shrub native to the Horn of Africa and the Middle East. The leaves of this small tree are chewed by millions worldwide as a mild, amphetamine-like psychostimulant drug. Whilst khat has enjoyed grey-area legality in many countries including the United Kingdom that otherwise prohibit the use of recreational drugs, recent years have seen the appearance of new legislation to control its production, importation and consumption. This mini-review will consolidate what is known about the pharmacology, chemistry, and effects on health of khat to provide a brief synopsis of the properties of this plant stimulant.     

Constipating and spasmolytic effects of Khat (Catha edulis Forsk) in experimental animals.

The constipating and spasmolytic effects of Catha edulis Forsk (Khat) were investigated in whole mice and on isolated guinea pig ileum. D-amphetamine was employed in both experiments for comparison. The total distance travelled (expressed in percentage) by charcoal suspension in the gastrointestinal tract of mice was determined before and after khat administration. The procedure was repeated with amphetamine and normal saline. The results were compared. Amplitudes of contraction were recorded with standard spasmogens, histamine and carbachol, in the presence and absence of khat extract of different concentrations. The same was done with amphetamine. Khat extract was observed to reduce the total distance travelled by charcoal suspension, comparable to D-amphetamine. The spasmogenic effects of both histamine and carbachol were observed to be antagonized by the khat extract in a concentration-dependent manner. The antispasmodic effect of khat extract was observed to be similar to that of D-amphetamine.     

Concentration Levels of Essential and Non-essential Metals in Ethiopian Khat (Catha edulis Forsk)

The levels of essential (Ca, Mg, Mn, Fe, Zn, Cr, Cu, and Co) and non-essential (Cd and Pb) metal in six different varieties of Ethiopian khat (Catha edulis Forsk, an evergreen stimulant plant) commonly consumed in the country and exported to the neighboring countries were determined by flame atomic absorption spectrometry. Known weight of oven-dried khat samples were wet-digested using 2 mL of (69–72%) HNO₃ and 2 mL of (70%) HClO₄ for 2 h and 30 min at variable temperature (120–270°C). The mineral contents in the digests were analyzed using flame atomic absorption spectrometer. The following concentrations ranges in fresh-weight basis were recorded in decreasing order: Ca (1,038–2,173 µg/g)?>?Mg (478.2–812.3 µg/g)?>?Fe (53.95–82.83 µg/g)?>?Zn (5.18–9.40 µg/g)?>?Mn (6.98–8.66 µg/g)?>?Cu (1.85–5.53 µg/g)?>?Cr (0.66–3.47 µg/g)?>?Co (0.41–0.80 µg/g). A wide variation in the mineral contents of khat from different region of Ethiopia was noticed. The toxic metals (Pb and Cd) were not detected in all the samples analyzed.     

Cathinone: An alkaloid of Catha edulis (Khat) exacerbated hyperglycemia in diabetes-induced rats

Cathinone, the main bioactive alkaloid of Catha edulis (khat), slightly increased the blood sugar levels of healthy animals, while its effect on blood sugar levels of diabetic animals has not yet been reported. This study investigated the in vitro inhibition of cathinone on α-amylase and α-glucosidase as well as its in vivo glycemic effects in diabetes-induced rats. Rats were fed on a high fat diet for five weeks, which then intraperitoneally injected with streptozotocin (30 mg/kg). Diabetic rats were distributed randomly into diabetic control (DC, n = 5), 10 mg/kg glibenclamide-treated group (DG, n = 5), and 1.6 mg/kg cathinone-treated group (CAD, n = 5). Additional healthy untreated rats (n = 5) served as a nondiabetic negative control group. Throughout the experiment, fasting blood sugar (FBS), caloric intake and body weight were recorded weekly. By the 28th day of treatment, rats were euthanized to obtain blood samples and pancreases. The results demonstrated that cathinone exerted a significantly less potent in vitro inhibition than α-acarbose against α-amylase and α-glucosidase. As compared to diabetic control group, cathinone significantly increased FBS of diabetic rats, while insulin levels of diabetic rats significantly decreased. In conclusion, cathinone was unable to induce a substantial in vitro inhibition on α-amylase and α-glucosidase, while it exacerbated the hyperglycemia of diabetes-induced rats.     

Ontogeny of the ovule and seed wing in Catha edulis (Vahl) Endl. (Celastraceae)

The seed wing of Catha edulis (Vahl) Endl. (Celastraceae) has been described as an aril derived from the funiculus. However, the aril-like structure of other Celastraceae has been shown to originate from the micropyle instead of the funiculus. It is therefore of interest whether the seed appendages in Catha are not only functionally but also developmentally different from those in other Celastraceae. To clarify this, the ontogeny of ovule and seed wing in Catha edulis has been studied. It is shown that the wing is a modified caruncula derived from the micropylar region. Seed wings are thus not only interesting in respect to function but may also carry interesting and important evolutionary signals. The evolution of different micropylar appendages within Celastraceae is discussed.     

Endophytic fungi associated with leaves of Betulaceae in Japan

Diversity and species composition of endophytic fungi on leaves of 11 tree species in Betulaceae were studied, with reference to climatic, tree species, and seasonal variations. A total of 186 fungal isolates were obtained from 190 leaves collected in a subalpine forest, a cool temperate forest, and a subtropical forest in Japan, and were divided into 46 operational taxonomic units (OTUs) according to the base sequences of D1-D2 region of large subunit rDNA. The 2 most frequent OTUs were Muscodor sp. and Nemania sp. in Xylariaceae, followed by Gnomonia sp., Glomerella acutata , Apiosporopsis sp., Asteroma sp., and Cladosporium cladosporioides . The similarities of OTU composition in endophytic fungal assemblages on leaves of Betulaceae were generally low among the forests of different climatic regions. Fungal OTU compositions were relatively similar between 2 Betula species in the subalpine forest, whereas 7 tree species in the cool temperate forest were divided into 3 groups according to the similarity of endophytic fungal assemblages on the leaves, with 4 Carpinus species assigned into 2 of the 3 groups. The similarity of endophytic fungal assemblages between August and October was relatively high in the subalpine forest, whereas the seasonal changes were generally greater (i.e., the similarities among sampling dates were lower) in the cool temperate forest.     

Cytotoxicity of Khat (Catha edulis) extract on cultured mammalian cells: effects on macromolecule biosynthesis

A chloroform extract of Khat (Catha edulis) leaves was used to study the cytotoxic activity on KB, 1BR.3, and XP2Bi cells. Log phase cell survival curves showed an LD50 of 40 ng/ml for KB cells. 1BR.3 and XP2Bi cells were biphasic in their response to the extract during log phase, with an LD50 of 20 and 75 ng/ml, respectively. Stationary phase cells were unaffected by the extract. DNA and RNA synthesis inhibition was studied using radiolabeled thymidine or uridine to measure the amount of extract that inhibits the synthesis to 50% of the untreated control cells. DNA synthesis was inhibited by 45, 60 and 200 ng/ml and RNA synthesis by 24, 17 and 58 ng/ml in 1BR.3, XP2Bi and KB cells, respectively. Protein synthesis was inhibited to 15-20% of untreated control cells by a dose of 40 ng/ml in all the cells studied. From this work, it is apparent that the main cause of cytotoxicity of Khat extract may be the inhibition of de novo RNA synthesis. Our results suggest that this effect is exerted on all cells used in this study and that KB cells demonstrate a higher resistance to the toxic component.     

Isolation of endophytic fungi from leaves of Pasania edulis and their within-leaf distributions

 Endophytic fungi were isolated from leaves of Pasania edulis, one of the most important trees of the warm temperate forests in southern Kyushu, by the surface sterilization method using H₂O₂ as a sterilizing agent. From a tree in the Experimental Nursery of Kagoshima University, located at the city of Kagoshima, Phyllosticta sp. and Colletotrichum spp. were frequently isolated. From a stand in a laurel forest in Mt. Takakuma, an ascomycetous fungus (Ascomycete sp. 1) and Phomopsis sp. were frequently isolated. Phyllosticta sp. was isolated more frequently from petiole segments and leaf segments with midrib and Phomopsis sp. from petiole segments and leaf-base segments with midrib than other segments. Colletotrichum spp. were isolated less frequently from petioles and Ascomycete sp. 1 from petiole segments and leaf-base segments with midrib than other segments. As possible causes of such biases in within-leaf distributions of the endophytes, differences in infection modes and negative interactions of major endophytes within leaves are suggested. Received: December 13, 2001 / Accepted: June 7, 2002 Acknowledgments The authors thank the staff members of the Experimental Forests of Kagoshima University for enabling the present study. Correspondence to:K. Hata     

Cossidae of the socotra archipelago (yemen)

The faunistic composition of the family Cossidae (Lepidoptera) of the Socotra Archipelago is revised. Five species are recognized, including two new species (Mormogystia brandstetteri and Meharia hackeri), and dubious identifications and records are discussed. Adults and genitalia are illustrated and bionomic details, DNA barcodes and a synonymic checklist for Socotran cossids are provided. A review of their distribution reveals that at least 80 percent of Socotra's cossids are unique to the archipelago, which is renowned for its endemism. A checklist listing all the species from generas Meharia, Mormogystia, Aethalopteryx, Azygophleps, as well as the synonymy and distribution is provided.     

Allometric equations for yield predictions of enset (Ensete ventricosum) and khat (Catha edulis) grown in home gardens of southern Ethiopia

Enset is a large, single‐stemmed perennial herbaceous plant domesticated as a staple food crop only in Ethiopia. Khat is a perennial plant cultivated for its economically important leaves and twigs that are the sources of stimulant when chewed. We address the issue of yield estimation of both crops, as they are important for the livelihoods of smallholders in the home garden systems in Southern Ethiopia and have received little attention so far. The objective of this study was to develop linear allometric models for estimating the edible (food and feed) and commercial yields of enset and khat plants, respectively. Data were collected from 20 enset and 100 khat plants. Diameter at 50‐cm height (d₅₀), pseudostem height (h_p) and their combination were good predictor variables for the food products of enset with adjusted R² values above 0.85, while d₅₀, h_p, edible pseudostem height (h_ep), total height (h_t) and their combination were good predictor variables for the feed products of enset with adjusted R² values above 0.70. For dwarf khat plants crown area (ca) combined with total height (h_t) resulted in the best prediction with an adjusted R² of 0.77, while the leaf and twig dry weight for tall khat plants was best predicted by ca with adjusted R² of 0.43. In all cases linear models were used.     

Ribotyping of vibrio populations associated with cultured oysters (Ostrea edulis)

The intraspecific variability of Vibrio splendidus, V. harveyi and V. tubiashii recovered from oysters (Ostrea edulis) collected at the Mediterranean coast near Valencia, Spain, was analyzed by ribotyping. The two former species represented the most abundant ones, and the third one was the only species described as pathogenic for oysters. A total of 115 environmental strains were studied, 84 of V. splendidus, 23 of V. harveyi and 8 of V. tubiashii. Chromosomal DNA was digested with KpnI and hybridized with an oligonucleotide probe complementary to a highly conserved sequence in the 23S rRNA gene. Ribotyping among natural populations of the three species rendered 5 to 9 bands, and showed a high genetic diversity, with a ratio no. of strains/no. of ribotypes between 1.1 and 1.5. Cluster analysis of V. splendidus ribotypes suggests a seasonal pattern of incidence, with those ribotypes corresponding to winter and spring samples being maintained in the oysters over the year.     

Transcriptome Profiling of Khat (Catha edulis) and Ephedra sinica Reveals Gene Candidates Potentially Involved in Amphetamine-Type Alkaloid Biosynthesis

Amphetamine analogues are produced by plants in the genus Ephedra and by khat (Catha edulis), and include the widely used decongestants and appetite suppressants (1S,2S)-pseudoephedrine and (1R,2S)-ephedrine. The production of these metabolites, which derive from L-phenylalanine, involves a multi-step pathway partially mapped out at the biochemical level using knowledge of benzoic acid metabolism established in other plants, and direct evidence using khat and Ephedra species as model systems. Despite the commercial importance of amphetamine-type alkaloids, only a single step in their biosynthesis has been elucidated at the molecular level. We have employed Illumina next-generation sequencing technology, paired with Trinity and Velvet-Oases assembly platforms, to establish data-mining frameworks for Ephedra sinica and khat plants. Sequence libraries representing a combined 200,000 unigenes were subjected to an annotation pipeline involving direct searches against public databases. Annotations included the assignment of Gene Ontology (GO) terms used to allocate unigenes to functional categories. As part of our functional genomics program aimed at novel gene discovery, the databases were mined for enzyme candidates putatively involved in alkaloid biosynthesis. Queries used for mining included enzymes with established roles in benzoic acid metabolism, as well as enzymes catalyzing reactions similar to those predicted for amphetamine alkaloid metabolism. Gene candidates were evaluated based on phylogenetic relationships, FPKM-based expression data, and mechanistic considerations. Establishment of expansive sequence resources is a critical step toward pathway characterization, a goal with both academic and industrial implications.