Coffee and tomato share common gene repertoires as revealed by deep sequencing of seed and cherry transcripts

An EST database has been generated for coffee based on sequences from approximately 47,000 cDNA clones derived from five different stages/tissues, with a special focus on developing seeds. When computationally assembled, these sequences correspond to 13,175 unigenes, which were analyzed with respect to functional annotation, expression profile and evolution. Compared with Arabidopsis, the coffee unigenes encode a higher proportion of proteins related to protein modification/turnover and metabolism—an observation that may explain the high diversity of metabolites found in coffee and related species. Several gene families were found to be either expanded or unique to coffee when compared with Arabidopsis. A high proportion of these families encode proteins assigned to functions related to disease resistance. Such families may have expanded and evolved rapidly under the intense pathogen pressure experienced by a tropical, perennial species like coffee. Finally, the coffee gene repertoire was compared with that of Arabidopsis and Solanaceous species (e.g. tomato). Unlike Arabidopsis, tomato has a nearly perfect gene-for-gene match with coffee. These results are consistent with the facts that coffee and tomato have a similar genome size, chromosome karyotype (tomato, n=12; coffee n=11) and chromosome architecture. Moreover, both belong to the Asterid I clade of dicot plant families. Thus, the biology of coffee (family Rubiacaeae) and tomato (family Solanaceae) may be united into one common network of shared discoveries, resources and information.
Introduction
Coffee is an important international commodity, ranking among the five most valuable agricultural exports from developing countries (Food and Agriculture Organization, http://apps.fao.org). Moreover, production and processing of coffee employs more than 25 million people worldwide (O’Brien and Kinnaird 2003). Despite its economic importance, coffee has received little attention with respect to molecular genetics and genomics research. As of December 2004, only 1,570 nucleotide and 115 protein sequences from coffee had been deposited in GenBank with the majority of those sequences derived from leaf ESTs. Many of the remaining sequences correspond to enzymes in the caffeine biosynthesis pathway—the most extensively studied pathway in coffee (Moisyadi et al. 1998; Ogawa et al. 2001; Mizuno et al. 2003; Uefuji et al. 2003).
Commercial coffee production relies mainly on two closely related species: Coffea arabica and Coffea canephora, accounting for approximately 70 and 30% of worldwide coffee production, respectively (Herrera et al. 2002). Although C. canephora accounts for a lower total proportion of the coffee market than does C. arabica, it is the main source for soluble coffee, which is consumed widely throughout the world. C. canephora is a diploid (2n=2x=22), outcrossing and highly polymorphic species native to central Africa, but which has expanded, through cultivation, especially to western Africa, Indonesia and Vietnam (Wrigley 1988). In contrast, C. arabica is believed to be a recently derived tetraploid (2n=4x=44) native to a small region of what is now Ethiopia. C. arabica is now grown widely throughout the world.
The goal of the current project was to increase the genetic and molecular knowledge of coffee through the generation and annotation of an EST database using high throughput single-pass 5′ sequencing of cDNAs derived from leaf, pericarp and seed tissues from a set of C. canephora varieties. Special emphasis was given to sequencing cDNAs from different stages of seed development, both to shed light on this important, but not well understood aspect of plant development and to capture as many genes as possible involved in determining the final chemical composition of seeds which constitute the commercial product. As a result, the EST database reported herein is, to our knowledge, the largest public database of seed-derived ESTs (White et al. 2000; Suh et al. 2003).
The EST database was used to derive a coffee unigene build, which was subsequently subjected to functional annotation. The coffee unigene build was compared with the model species Arabidopsis. Arabidopsis is the only fully sequenced dicot genome and hence provides a complete set of predicted genes against which to compare the EST-derived unigene set of coffee. However, coffee and Arabidopsis belong to different plant families (Rubiaceae and Brassicaceae, respectively) which are distantly related phylogenetically and which diverged from their last common ancestor approximately 94 million years ago (MYA) (Gandolfo et al. 1998; Crepet et al. 2004) (Fig. 1).
Fig. 1Dendrogram depicting phylogenetic relationships of coffee to other higher plant taxa (based on Chase et al. 1993)
The plant family most closely related to coffee in which extensive sequencing has been conducted is Solanaceae (Fig. 1). In this family, comprehensive EST databases have been developed for tomato, potato, pepper, eggplant and petunia (http://www.sgn.cornell.edu/) (Hoeven et al. 2002; Ronning and Stegalkina 2003; Lee et al. 2004). Both Rubiaceae and Solanaceae belong to the Asterid I clade of dicots, and based on existing fossil evidence, are thought to have diverged from one another approximately 50 MYA (Gandolfo et al. 1998; Crepet et al. 2004) (Crepet personal communication) (Fig. 1). The closer taxonomic affinities of coffee and Solanaceae (e.g. tomato) are paralleled by a number of striking botanic and genetic similarities, including the production of fleshy berries, a similar genome content (C=950 and 640 Mb for tomato and coffee, respectively) (Hoeven et al. 2002), similar basic chromosome number (x=12 for tomato and most other Solanaceae; x=11 for coffee) and similar chromosome architecture with highly condensed pericentric heterochromatin and decondensed euchromatin at the pachytene stage of meiosis (Rick 1971; Pinto-Maglio and Cruz 1998). For these reasons, the coffee unigene set was also compared against a series of Solanaceae EST-derived unigene sets.
Materials and methods
Library construction
Source of tissues
C. canephora was farm-grown in east Java. Plant tissue was frozen in liquid nitrogen, transported on dry ice and stored at –80°C until RNA extraction. The specified tissues (leaf, pericarp, young cherries with seed and pericarp mixed, middle stage seed and late stage seed) were collected from five different varieties Table 1). The maturation period of the C. canephora varieties under study is approximately 9–11 months, from pollination to ripening. The early stage cherry, middle stage seed and late stage seed were collected between 18 to 22 weeks, 30 weeks and 42 to 46 weeks after pollination, respectively.
Table 1Characteristics of the 5 cDNA libraries used to develop the coffee EST databaseLibrary nameTissueVarietiesAverage insert size, kbGood quality ESTsLeafLeaves, youngBP4091.5±0.68,942PericarpPericarp, all developmental stagesBP358, BP409, BP42, BP961, Q1211.4±0.58,956Early stage cherryWhole cherries, 18 and 22 week after pollinationBP358, BP409, BP42, Q1211.4±0.39,843Middle stage seedEndosperm and perisperm of seeds, 30 week after pollinationBP409, BP961, Q1211.4±0.310,077Late stage seedEndosperm and perisperm of seeds, 42 and 46 week after pollinationBP358, BP409, BP42, BP961, Q1211.4±0.39,096
RNA and mRNA isolation
Total RNA was extracted using phenol/chloroform (Rogers et al. 1999) and further treated with DNase I (RNase-free) and purified using an RNeasy Kit (Qiagen, Valencia, CA 91355). Messenger RNA was extracted from total RNA with PolyTrack mRNA Isolation Systems (Promega, Madison, WI 53711).
cDNA libraries
Directional cDNA libraries were constructed with 3–5 μg of mRNA with the ZAP-cDNA Gigapack III Gold Cloning Kit (Stratagene, La Jolla, CA 92037). The average insert length was estimated by PCR in 36 randomly selected clones from each library and ranged 1.2–1.5 kb (Table 1).
Sequencing
Bacteria, containing coffee cDNAs, were cultured in 384-well plates and cDNA inserts subjected to 5′ end sequencing at the BioResource Center at Cornell University (http://www.brc.cornell.edu). The average size of quality reads was 613 bp with a maximum of 1,037 bp.
Sequence quality processing
EST sequences were base-called and screened for vector sequences using PHRED software (Ewing et al. 1998). The longest stretch of overall high quality (PHRED score over 15 which corresponds to over 98% confidence) of each sequence was identified. PolyA repeats were trimmed to at most 20 bp and any sequence past the PolyA (mostly low quality sequence) was discarded. After the trimming, the sequences were screened against the E.coli K12 genome to remove any bacteria contamination. The remaining sequences were screened for minimum length (150 bp) and maximum allowed ambiguity (4%) and low complexity (60% of the sequence are of the same nucleotide, or 80% of same two nucleotides, which indicate error in sequencing).
Unigene assembly
Unigene sets were built by combining the sequences from all five coffee cDNA libraries. Clustering was performed using a program developed at the Sol Genomics Network (SGN at http://www.sgn.cornell.edu), which relied on a custom pre-clustering algorithm, and on the CAP3 program for contig generation (Huang and Madan 1999). The preclustering algorithm clustered sequences using a Smith Waterman type algorithm with initial word matching. The command line settings for CAP3 were as follows: -e 5000 -p 90 -d 10000 -b 60. The -e, -d and -b options are set such that the assembler disregards them or minimizes their effect. The -p option increases the sequence identity necessary on overlaps to 90 from a default of 75, which were found to be not stringent enough. Sequences were also checked for length, complexity and contamination. The builds were uploaded to the database, where each unigene was assigned a unique unigene ID.
Annotation
Protein prediction
The most likely coding frame and the corresponding transcribed peptide for each unigene were generated using ESTScan, a program that detects coding regions in EST sequences and corrects some sequencing errors (nucleotide substitution, deletion/insertion, erroneous stop codon, etc) (Iseli et al. 1999). As a hidden Markov Model (HMM) based program, ESTScan requires a training set of high quality coding sequences to generate a model. However, very few high quality coffee coding sequences are publicly available (less than 50 full length in GenBank). Therefore, we used as a training set of 483 nuclear genes from the closely related species, tomato (http://www.ebi.ac.uk/embl). Table 2 compares the coffee unigene assembly and translation (using ESTScan) with that of tomato, using the same training tomato set and parameters. The fact that the two give such similar results, gives confidence that the coffee EST-derived unigenes are being efficiently translated via the ESTScan software using the tomato training set (Table 2).
Table 2Comparison of the coffee and tomato EST databases derived from use of ESTScan calibrated with the same tomato training set (see Materials and methods for details)TomatoCoffeeTotal unigenes30,57613,175Average unigene length, bp774678Unigenes with coding regions96%95%Average length (bp) of predicated peptides569556Average ESTScan score409346
BLAST matches between coffee unigenes and other sequence databases
Sequence matches for coffee unigenes against other sequence databases were obtained using BLAST (Altschul et al. 1997) (see Results and discussion section for details of each BLAST analysis). The target databases were:The GenBank non-redundant (NR) protein and dbest dataset (NCBI, http://www.ncbi.nlm.nih.gov).The predicted Arabidopsis proteome and cDNA databases which were downloaded from the Arabidopsis Information Resource (TAIR, http://www.arabidopsis.org and ftp://ftp.arabidopsis.org/home/tair/).The Solanaceae EST-derived unigene sets, including tomato (Solanum. lycopersicum) (184,860 ESTs, 30,576 unigenes), potato (Solanum. tuberosum) (97,425 ESTs, 24,932 unigenes), pepper (Capsicum annuum) (20,738 ESTs, 9,554 unigenes), petunia (Petunia hybrida) (3,181 ESTs, 1,841 unigenes) and eggplant (Solanum melongena) (11,479 ESTs, 5,135 unigenes), all of which can be accessed at SGN (http://www.sgn.cornell.edu). To estimate ribosomal, chloroplast and mitochondrial contamination, the coffee unigene set was also screened against the Arabidopsis complete mitochondrial genome (NC_001284), tobacco ribosomal rRNA gene (5.8s AJ012367, 18s AJ236016 and 25s S52185) and the complete tobacco chloroplast genome (Z00044) sequences from GenBank.
Functional annotation based on predicted peptides
ESTScan-predicted coffee peptides were subjected to InterPro Scan annotation, which integrates the most commonly used protein signature databases (PROSITE, PRINTS, Pfam, ProDom, etc.) together with their associated scanning methods for protein domain analysis (Apweiler et al. 2001; Zdobnov and Apweiler 2001). Based on the domain annotation, GO accession of the unigenes were assigned using interpro2go conversion file from the GO consortium (http://www.geneontology.org, also available at http://www.ebi.ac.uk/interpro).
Functional categorization based on gene ontology
GO annotations were formatted for input into the GOSlim program and the output parsed to count the occurrence of each GO category. GOSlims are ‘slimmed down’ versions of the ontologies that allow a high-level view of gene functions. The GOSlim file and program were obtained from the Gene Ontology Consortium at http://www.geneontology.org.
Gene family analysis
The predicted protein sequences for the coffee unigene set and the Arabidopsis protein set were combined into a single file, formatted as a blast database using formatdb, and run with BLASTP (protein vs. protein sequence) against itself with option m8 for output. The resulting file was used as the input for the tribeML program. TribeML (Enright et al. 2002) formats the clusters, such that each cluster was given in a tab delimited file, one cluster per line. Simple scripts were used to parse the information to detect the largest gene families, coffee specific families, and families that showed large expansions in coffee.
Results and discussion
Generation of coffee EST database and unigene set
A total of 62,829 cDNA clones, derived from mRNA from five different tissues, were subjected to 5′ sequencing. After quality evaluation (see Materials and methods for details), the database was reduced to 46,914 high quality ESTs, averaging 613 bp in length. This corresponded to approximately 9,000 high quality ESTs from each of the five cDNA libraries (Table 1). These high quality ESTs were assembled into 13,175 unigenes with average length of 678 bp (ranging 150–2,714 bp), among which 7,272 (55%) were singletons and 5,903 (45%) were contigs (Fig. 2). Of the contigs, the majority (87%) was represented by two to ten ESTs while some (13%) were comprised of more than ten ESTs (Fig. 2). All trace files, sequences and derived unigenes/annotation can be found at the Solanaceae Genomics Network (SGN) website (http://www.sgn.cornell.edu) and will be submitted to GenBank at the time of publication.
Fig. 2Histogram depicting the distribution of EST content for all coffee unigenes. Numbers above bars equals the number of unigenes represented in each
Differentiating between paralogs and alleles
The coffee ESTs were derived from five C. canephora varieties (Table 1), however, unigene assembly for EST reads does not automatically distinguish between polymorphic alleles and highly similar paralogs. C. canephora is known to be a highly allogamous, heterozygous and polymorphic species, raising the possibility that some of the EST-derived unigenes might actually represent different allelic forms of the same gene. In order to evaluate how well paralogs were distinguished from allelic polymorphism in the unigene assembly, BLASTN (nucleotide vs. nucleotide sequences) was performed amongst all of the coffee unigenes and the results parsed to identify the best non-self matches. As a control, BLASTN was performed in a similar manner on a complete set of Arabidopsis predicted genes, including both coding and UTR regions obtained from TAIR (http://www.arabidopsis.org). Since the Arabidopsis database was derived from the complete genome sequence of a single, inbred stock (Columbia), the entire gene complement is present without the complication of allelic variation (initiative). For both coffee and Arabidopsis, pair-wise combinations for which the homology extended over more than 70% of the sequence were recorded. The sequence similarity for each of these combinations was plotted for both coffee and Arabidopsis (Fig. 3).
Fig. 3Plot depicting the sequence identify of the most similar match for each coffee unigene as compared with all other coffee unigenes. As a control, a similar analysis is shown for Arabidopsis genes (see Results for details)
The Arabidopsis plot showed two peaks, one with low identity (~87%) and the other with higher identity (over 99%) with 6.4 and 1.6% of genes falling into the low and high identity peaks, respectively. Like Arabidopsis, the coffee plot also showed two peaks, one with lower identity (around 91%) and the other with high identity (over 99%). These two peaks corresponded to 6.2 and 0.8% of the total unigenes, respectively. ESTs corresponding to ten pairs of coffee unigenes, from the >99% peak, were used as probes on genomic southern hybridizations to determine whether the matching pairs were truly duplicated in the coffee genome (paralogs) or rather allelic (single copy). For eight of the ten pairs, the paired ESTs hybridized the same single copy gene on southerns (data not show). Thus, a significant number (approximately 80%) of the unigenes in this peak are likely to be allelic. However, this category represented only a small portion of the coffee unigenes (0.8%). A similar experiment was performed with 11 ESTs from 11 pairs of unigenes in the second, lower homology peak (around 91% identity, see Fig. 3). In this case, the majority (8 out of 11) was determined to represent true paralogs (two or more copies in the genome) (data not shown). Thus for further discussions, it is assumed that the majority coffee EST-derived unigenes do in fact correspond to unique coffee genes.
Functional annotation of coffee EST-derived unigenes
Predicted coffee proteins
ESTScan (see Materials and methods for details) was able to identify protein-coding sequences in 12,534 coffee unigenes (95% of total unigenes), among which 1,515 (12%) were putatively full-length (starting with ATG and ending with a stop codon). Due to the cDNA library construction method, the unigenes were biased for the 3′ end—57% of the unigenes covered the 3′ end (ending with a stop codon) while only 36% covered the 5′ end (starting with ATG). Of the 5% of unigenes from which a protein sequence could not be predicted, 81% were singletons and the majority (97%) did not match to any Arabidopsis, GenBank non-redundant (NR) or Solanaceae unigene sequences, suggesting that they are not bona fide gene transcripts.
Protein domain annotation
Peptides translated from ESTScan predicted coding sequences were subjected to InterPro Scan (see Materials and methods for details) for protein domain analysis. A total of 1,678 distinct domains were identified in the database, which were present in 4,414 (35%) unigenes. Table 3 lists the 20 most represented InterPro domains found in the coffee genes, as well as comparative statistics for tomato and Arabidopsis genes. Coffee and tomato were very similar with regard to the most frequent protein domains; however, a number of striking differences were noted when comparing coffee and Arabidopsis. Most notably, the following five domains were much more common in coffee encoded proteins than in Arabidopsis: proline-rich regions, tyrosine protein kinases, glucose/ribitol dehydrogenases, ubiquitins and ubiquitin-conjugating enzymes. The latter two categories, involving ubiquitinization, suggest a more active and/or complex system for protein turnover in coffee versus Arabidopsis.
Table 3Twenty most abundant InterPro domains identified in coffee unigene set and comparative statistics for tomato and Arabidopsis genesInterPro accessionDescription% of unigenes (ranking)CoffeeTomato
Arabidopsis
IPR000719Protein kinase1.61.20 (1)3.0 (1)IPR000694Proline-rich region1.30.91 (4)0.003 (1763)IPR002290Serine/threonine protein kinase0.851.10 (2)0IPR001245Tyrosine protein kinase0.691.0 (3)0.15 (311)IPR008271Serine/threonine protein kinase, active site0.610.68 (5)2.6 (2)IPR000504RNA-binding region RNP-1 (RNA recognition motif)0.550.60 (6)0.59 (6)IPR001680G-protein beta WD-40 repeat0.490.51 (8)0.51 (8)IPR001611Leucine-rich repeat0.480.59 (7)0.59 (7)IPR002048Calcium-binding EF-hand0.360.34 (13)0.34 (13)IPR000379Esterase/lipase/thioesterase0.330.43 (10)0.43 (10)IPR001806Ras GTPase superfamily0.320.26 (22)0.43 (70)IPR003579Ras small GTPase, Rab type0.290.23 (27)0IPR0051232OG-Fe(II) oxygenase superfamily0.270.26 (21)0.47 (52)IPR000626Ubiquitin0.270.22 (32)0.40 (89)IPR002401E-class P450, group I0.270.46 (8)0.77 (24)IPR002347Glucose/ribitol dehydrogenase0.260.23 (28)0.33 (110)IPR001005Myb DNA-binding domain0.260.34 (15)1.34 (8)IPR005225Small GTP-binding protein domain0.260.24 (25)0.68 (27)IPR000608Ubiquitin-conjugating enzymes0.260.21 (34)0.19 (221)IPR007090Leucine-rich repeat, plant specific0.250.40 (12)1.07 (11)
Gene ontology annotation
Gene ontology (GO) annotations were made for each coffee unigene based on InterPro domain annotation (see Materials and methods for details). A total of 3,248 unigenes (25% of total unigenes and 74% of unigenes with InterPro domain match) could be assigned a GO annotation. In order to obtain an overview of representation of biological processes by the coffee unigene set, the GO annotations were mapped to the plant GOSlim, a group of selected higher-level categories of the GO ontology (see Materials and methods for details). Figure 4 depicts the biological process GOSlim categories for each of which at least 1% of the coffee unigenes were assigned. Metabolism is the most abundant category, both in terms of number of subcategories (14 subgroups) and number of unigenes in the subcategories (2,541 unigenes and ~80% of the GO annotated unigenes). In addition, signal transduction, cell growth and/or maintenance, response to stress and response to endogenous stimulus were among the most abundant categories.
Fig. 4Comparison of the gene ontology-based gene annotation categories for the coffee EST-derived unigene set, tomato EST-derived unigene set and the Arabidopsis proteome. Figure contains only categories in which more than 1% of the coffee unigenes were assigned. Categories for which coffee differs most significantly from Arabidopsis are shown in underline bold. (1) Cellular processes other than signal transduction and cell growth and/or maintenance. (2) Nucleobase/nucleoside/nucleotide and nucleic acid metabolism other than DNA metabolism and transcription. (3) Protein metabolism other than protein biosynthesis and protein modification. (4) Metabolism other than amino acid and derivative metabolism, biosynthesis, carbohydrate metabolism, catabolism, electron transport, lipid metabolism, nucleobase/nucleoside/nucleotide and nucleic acid metabolism and protein metabolism. (5) Cell growth and/or maintenance other than cell cycle and cell organization and biogenesis. (6) Physiological processes other than photosynthesis, response to stress, response to endogenous stimulus, response to external stimulus and metabolism
A comparison was made between the GOSlim biological process of Arabidopsis, tomato and coffee (Fig. 4). For both the tomato and coffee unigene sets, the GO annotations were based on InterProScan results and approximately 25% of both unigene sets were assigned a GO annotation. In Arabidopsis, the genes are of full length, giving a higher chance of finding functional domains. Moreover, extensive experimental research and manual annotation has been carried out in Arabidopsis, resulting in a higher proportion of genes with assigned GO annotation. Therefore 83% of the Arabidopsis genes are assigned GO annotations. No significant differences were observed in the annotated categories for coffee versus tomato—possibly reflecting their close taxonomic affinity. However, for a number of categories, coffee had significantly different proportions of genes than Arabidopsis. The categories with the largest significant differences (P<0.001, based on Chi-square test) are: carbohydrate metabolism, other metabolism, biosynthesis, catabolism, protein biosynthesis, protein modification and energy pathways. In all cases, coffee had a significantly higher proportion of genes in these categories than Arabidopsis (Fig. 4). It is interesting to note that many of these categories center around the synthesis, breakdown or modification of compounds. One of the hallmarks of coffee is its high-level diversity of primary and secondary compounds, which contributes to the sensory quality of brewed coffee beans. The Rubiaceae family in general contains some of the most diverse species with regard to secondary metabolism and is an especially rich source of alkaloids—a number of which have pharmacological and/or psychotropic properties (Kutchan 1995; Facchini 2001). In fact, the most widely used psychotropic drug, caffeine, comes mainly from coffee. One can speculate that this metabolic diversity is reflected in the relatively high proportion of coffee genes with putative functions related to metabolism.
In silico analysis of unigene expression
Complexity and uniqueness of different stages/tissues
In an effort to determine which tissues gave rise to the more redundant or more complex pools of gene expression, three library/tissue parameters were calculated: (1) the percentage of total coffee unigenes represented by each library/tissue; (2) the percentage of more highly expressed unigenes (represented by ten or more ESTs from the library) in each library/tissue; (3) the percentage of unigenes from each tissue/stage which are unique to that library/tissue (not found in any other cDNA library). The results from these calculations are depicted in Fig. 5. The pericarp and leaf libraries represented more unigenes, contributed to more unique unigenes, and contained fewer highly abundant unigenes than did the early cherry, middle seed and late seed libraries—perhaps reflecting the overall lower complexity of these latter stages/tissues (Fig. 5). Among the seed stages, the middle stage of development gave more novel genes than did the early or late stages of seed development (Fig. 5).
Fig. 5Characteristics of each coffee cDNA library in comparison to the entire coffee EST-derived unigene set. The total unigene and highly expressed unigene categories sum to greater 100% since the same unigene may contain ESTs from more than one library
Differential expression of genes across stages/tissues
The coffee cDNA libraries were non-normalized and deeply sampled, and thus could be used to compare gene expression amongst the five cDNA libraries using statistical method (Audic and Claverie 1997). In all the pair-wise comparison between the cDNA libraries, only a small proportion of the overall unigenes (approximately 5%) showed significantly different (P<0.05) expression levels between two or more libraries (Table 4). The leaf and the early stage cherry libraries showed the highest divergence in gene expression (752 unigenes which is 5.7% of total unigenes, showed significant differential expression) while the leaf and the pericarp libraries had the lowest number of differentially expressed unigenes (384 unigenes and 2.9% of total). Interestingly, the three stages of seed development showed as much differential expression, relative to each other, as they did to non-seed tissues (leaf and pericarp). These results provide strong evidence that throughout the approximately 11 months from pollination to mature seed, a continuously changing cascade of molecular events is unfolding in the developing seed.
Table 4Number of coffee unigenes showing significantly (P<0.05) different expression in pairwise comparisons of cDNA librariesLibraryPericarpEarly stage cherryMiddle stage seedLate stage seedLeaf384752548562Pericarp610458527Early stage cherry602728Middle stage seed585
Highly expressed genes
Table 5 lists the blast annotation, library composition and the best Arabidopsis/Solanaceae gene match for the 20 most highly expressed unigenes from the coffee EST database. Some of these highly expressed genes are common to and highly expressed in all plant species—a good example of this is Unigene 122071, which encodes the small subunit of ribulose bisphosphate carboxylase—a key enzyme in photosynthesis and carbon fixation (Table 5). However, a number of these highly expressed genes are unique to coffee, and many have expression patterns confined to particular tissue/stages, providing a glimpse into some of the potential unique aspects of coffee biology. A brief description of some of these is given below.
Table 5The 20 most highly expressed coffee unigenes: functional annotation and most similar Arabidopsis and Solanaceae homologsCoffee unigene#: annotationBest match (e value/score)EST countArbidopsisSolanaceae Unigene_speciesTotal ESTsLeafPericarpEarly stage cherryMiddle stage seedLate stage seed125230: putative 2s seed storage proteinND243065_tomato (e-103/238)1,219
7
15
21
1,037
139
120912: 11s seed storage proteinAt5g44120 (1e-88/324)228376_tomato (0/802)687
0
3
28
244
412
121707: unknown functionAt1g29050 (1e-139/489)246695_potato (e-163/283)324
2
3
1
149
169
120118: unknown functionAt5g59320 (2e-21/99.8)221585_tomato (e-134/475)292
0
0
3
58
231
124988: unknown functionNDND20458
84
55
1
6
120685: chitinaseAt5g24090 (2e-43/172)214596_tomato (1e-35/84.5)202
99
40
58
0
5
124158: photoassimilate-responsive proteinAt3g54040 (2e-36/149)196924_pepper (2e-39/138)182
1
1
2
150
28119890: unknown functionND204426_pepper (5e-07/52.8)183
0
0
0
183
0123265: ADP-ribosylation factorAt2g47170 (1e-99/359)238338_tomato (0/693)18258
14
65
21
24
124083: secretory peroxidaseAt4g21960 (e-153/537)196145_pepper (0/681)161
55
5
19
49
33
124911: metallothioneinAt5g02380 (0.32/32.3)207464_petunia (2e-06/51.0)163
40
65
30
11
17
119817: chitinaseAt3g12500 (e-103/373)248120_potato (e-148/521)148
0
22
0
0
126
124815: unknown functionAt3g29240 (1e-87/320)227940_tomato (e-146/517)145
2
0
1
0
142
122206: SAM synthaseAt2g36880 (0/711)270415_petunia (0/887)142
1
9
130
0
2
119460: WRKY4 transcription factorAt1g80840 (3e-75/279)237166_tomato (e-137/487)123
0
0
123
0
0
123045: unknown functionAt3g16000 (0.69/31.2)218824_tomato (90.36/33.1)123
81
17
22
3
0
120481: AdoMet synthaseAt4g01850 (0/723)243236_potato (0/886)108
19
27
31
20
11
121265: Mobl/phoceinAt5g45550 (e-119/425)196814_pepper (e-146/513)113
1
0
112
0
0
124791: plasmodesmal receptorAt5g15140 (1e-99/360)203764_pepper (8e-86/314)105
0
0
3
26
76122071: rubiso small subunitAt1g67090 (9e-70/260)207453_petunia (3e-89/297)99
76
8
9
6
0
BLAST match values are given in parenthesesBold numbers indicate library for which the highest number of ESTs were observed for each gene. Italic numbers indicate library for which the number of EST is significantly lower (P<0.05) than the highest
Seed storage protein genes
Unigene 125230: a putative 2S seed storage protein
Unigene 125230 is the most highly expressed gene across the entire coffee unigene set (1219 ESTs) and was the dominant transcript in the middle stage seed library, accounting for 10% of the ESTs at this stage (Table 5). This gene shows high homology to a tomato unigene derived from a developing seed cDNA library, but has no detectable homolog in Arabidopsis (Table 5). Other than the match with tomato, weak homology was also detected for 2S seed storage proteins from sesame, sunflower, and Brazil nut (in decreasing order of similarity). It is interesting to note that coffee, tomato, sesame and sunflower are fairly closely related taxonomically. All belong to the Asterid I/II clade of Eudicots (Fig. 1). This close phylogenetic relationship may explain why Unigene 125230 has homologous matches only in these species. Moreover, since Unigene 125230 shows homology to the 2S seed storage proteins in these related species, we conjecture that unigene 125230, its tomato unigene match, the sesame, Brazil nut and sunflower 2S storage protein gene all encode orthologous 2S seed storage proteins. This is the first time that a 2S seed storage protein has been identified in coffee or any Solanaceae species. Finally, a BLAST search of Unigene 125230 against the coffee unigene set revealed additional putative copies of the 2S seed storage protein. However, on close examination, all appear to be splicing variants or low quality sequences. Moreover, southern hybridization with a 2S cDNA probe on genomic DNA confirmed that the 2S gene is single copy in the coffee genome (data not shown).
Unigene 120912: 11S seed storage protein
Unigene 120912 is the second most abundant unigene, containing 687 ESTs (Table 5). This gene is preferentially expressed during middle and late stage seed development and shares high similarity (over 98% identity) with a previously cloned C. arabica 11S seed storage protein (Marraccini et al. 1999; Rogers et al. 1999). This unigene also has a highly significant match to the Arabidopsis 12S storage protein and to a tomato unigene derived from seed ESTs (Pang et al. 1988) (Table 5). Given these results, we conclude that unigene 12912 is allelic with the previously described 11S seed storage protein gene from C. arabica and orthologous to 11S/12S seed storage proteins in both tomato and Arabidopsis. A BLAST search of Unigene 120912 against the coffee unigene set revealed additional putative copies of the 11S seed storage protein. However, like the 2s seed storage protein (Unigene 124230), all appears to be results of alternative splicing or low sequence quality. Moreover, southern hybridization with an 11S cDNA probe on genomic DNA confirmed that the 11S gene is single copy in the coffee genome (data not shown).
Other seed-specific genes
Early stage seed development
Unigenes 122206, 119460 and 121265 were all highly expressed and specific to the early cherry stage. The early cherry library was derived from RNA from both pericarp and seed tissue while the pericarp library was derived from RNA coming from all stages of pericarp development. Thus, if the above genes were highly expressed in the pericarp of the early cherry, they should be present in the pericarp library as well. The fact that these genes showed little or no expression outside the early cherry stage, suggests that they are probably specific to early developing seed tissues and not pericarp tissues (Table 5). Unigene 122206 showed high homology to an Arabidopsis gene annotated as encoding the enzyme S-adenosyl-l-methionine (SAM) synthetase (Table 5). This enzyme synthesizes S-adenosyl-l-methionine from l-methionine and ATP and is often represented by multiple isozymes in plant species (Schroder et al. 1997). Thus, Unigene 122206 appears to be an SAM synthetase specific to early seed development (Table 5).
Unigene 119460 shows high homology to the highly conserved WRKY transcription factor family. The WRKY transcription factor is a large gene family having more than 70 members in the Arabidopsis genome (Dong et al. 2003). Previous studies showed that it is related to wounding, stress, pathogen infection and senescence in many plant species. In some recent studies, the WRKY protein family was found to be involved in sugar signaling in barley and seed development in Arabidopsis (Johnson et al. 2002; Sun et al. 2003). However, the function of the best Arabidopsis match to Unigene 119460 (At1g80840) has not been determined. Hence, understanding the function of this highly expressed, WRKY-like coffee gene awaits further study.
Unigene 121265 is highly homologous to a gene in Arabidopsis annotated as encoding a Mob1/phocein protein. Mob1/phocein proteins are found in virtually all eukaryotes. While they are conjectured to be involved in cell cycle control, there is still little experimental evidence demonstrating biological function (Pon 2004). Thus it seems premature to conjecture what role Unigene 121265 might have that is specific to the early development of coffee seeds.
Middle stage seed development
As described earlier, Unigene 125230 is a putative 2S seed storage protein with peak expression during middle seed development. Also showing preferential expression during this same stage were Unigenes 121707, 124158 and 119890. Unigene 121707 is a gene of unknown function with high homology matches both in Arabidopsis and Solanaceae EST-derived unigenes (Table 5). Unigene 124158 is homologous to an Arabidopsis gene classified as a photoassimilate-responsive protein, which is related to pathogenesis (Herbers 1995). Finally, Unigene 119890, which is also specific to middle stage seed development, is apparently a gene unique to coffee, which will be discussed more in the following section.
Late stage seed development
As discussed previously, Unigene 120912 corresponds to the 11S seed storage protein, which is largely expressed late in seed development. Other genes with preferential expression in late stage seeds are Unigenes 120118, 119817 and 124791. Unigene 119817 likely encodes a chitinase and is further discussed in the next section. Unigene 120118 shows high homology to genes in both Arabidopsis and Solanaceae EST-derived unigene sets; however, none have known function. Unigene 124791 gives a strong match to an Arabidopsis gene annotated as a plasmodesmatal receptor.
Two highly expressed genes with homology to chitinase
Unigenes 120685 and 119817 show high sequence similarity to a number of genes classified as chitinases in other organisms. Chitinases are a large and diverse class of proteins, some of which have been implicated in resistance to fungi in various plant species, including coffee (Rojas-Herrera 2002; Chen et al. 2003). The two unigenes differ in that Unigene 120685 is expressed in leaves, pericarp and early stage cherries, but not in mid or late stage seed development (Table 5). Unigene 119817, on the other hand, was found to be exclusively expressed in late stage developing seeds and pericarp tissue. As previously mentioned, early stage cherries contained both pericarp and seed tissues. The fact that Unigene 120685 was not found in the middle and late stages of seed development suggests that this gene may not be expressed in seeds, but rather in the maternally derived pericarp and leaf tissues. Based on these results, one can speculate that these two putative chitinase genes may be involved in pathogen defense in developing CHERRIES, with Unigene 120685 being expressed in early developing, post pollination pericarp and leaf tissues and Unigene 119817 being expressed primarily late in seed development, just prior to maturity.
Highly expressed genes unique to coffee
Unigene 124988
This highly expressed unigene had no significant matches in the Arabidopsis proteome, Solanaceae EST-derived databases, GenBank NR databases, or GenBank dbest. Moreover, the predicted protein encoded by Unigene 124988 has no recognizable domains, which might give clues to its function. ESTs for this unigene were detected in all five libraries, with highest expression being observed in the pericarp (Table 5).
Unigene 119890
Unigene 119890 also has no significant match in any of the tested databases, with the possible exception of a very weak match in the Solanaceae unigene sets (the best hit was from pepper with an e value of 5e-7, Table 5). Like Unigene 124988, its predicted protein has no recognizable domains. Unigene 119890 was highly and exclusively expressed in the middle stage of developing seeds (Table 5).
The fact that neither Unigene 124988 nor Unigene 119890 have counterparts in any other databases suggests that they may represent coffee-specific genes or genes that have been evolving at such a rapid rate that they no longer bear any recognizable homologies with proteins from other plants, including the closely related Solanaceous plants. We speculate that these genes may be related to chemical or morphological features unique to coffee.
Gene families unique or significantly expanded in coffee
The predicted protein sequences for the coffee unigene set and the Arabidopsis proteome were combined and organized into protein families using tribeML software (see Materials and methods for details). In most cases, the number of gene family members was higher in arabidopsis than in coffee (data not shown). However, this is to be expected since the entire Arabidopsis genome has been sequenced (thus revealing all genes), whereas in coffee the EST-derived unigene set is by nature incomplete and unlikely to contain all members of any given gene family. However, despite the incomplete nature of the coffee EST-derived unigene set, several cases was encountered in which coffee has more gene family members than does Arabidopsis. To further investigate this topic, a list was made of the gene families for which coffee has significantly more members than Arabidopsis (Table 6). We refer to these as coffee-expanded gene families. In addition, gene families found in coffee, but for which no counterparts exist in Arabidopsis were also compiled (Table 7). We refer to these as coffee-unique gene families. All gene families listed in both Tables 6 and 7 were subjected to manual examination to remove any members determined to be results of alternative splicing and/or poor sequence quality—both of which could inflate the number of putative gene family members in coffee. Hence, the differences in copy number reported are likely to be due to true differences in gene copy number and not artifacts of EST-based gene predictions.
Table 6Gene families expanded in coffee relative to Arabidopsis
Family ## Arabidopsis family member# Coffee family memberLongest coffee memberAnnotation266121122330Retrotransposon gag protein, class I180514124952Polygalacturonase isoenzyme 1 beta subunit with BURP domain632112123451Acidic endochitinase386210124158Photoassimilate-responsive protein38248119672Hypersensitive-induced protein, band 7 protein39427122791E-class P45048326120054Bet v I allergen62336119581Root hair defective protein1,18215126674Unknown function69525126974Tyrosine decarboxylase78325122423Unknown function1,11725119449Trypsin inhibitor KunitzTable 7Gene families unique to coffee in comparison to Arabidopsis
Gene family ## Family memberLongest memberSolanaceae hitAnnotation24327122956258190 potatoRetrotransposon gag protein, class II68711120121221585 tomatoThaumatin, pathogenesis related96510119718249401 potatoZn-finger, CCHC type974101202442610402 potatoDisease resistance protein (TIR-NBS-LRR class)8529119638225732 tomatoRetrotransposon gag protein, classs III360812199823671 tomatoDisease resistance protein1,0197124574222350 tomatoLeucine-rich repeat, disease resistance protein1,6077122216noneUnknown function1,6107130519noneUnknown function1,6767126264243065 tomatoUnknown function7086123769236157 tomatoABA/WDS induced protein1,8525120284213688 tomatoProline-rich region, extension-like protein2,3625122218237314 tomatoUnknown function2,4595124466267984 potatoLeucine-rich repeat, plant specific, receptor-related protein kinase
Coffee-expanded gene families
The most expanded gene family in coffee corresponds to a retrotransposon gag protein (Table 6). This result has two implications. First, the retroelement encoding this gag protein occurs at a higher frequency in coffee compared with Arabidopsis, although we cannot determine whether this difference is due to a true expansion of this element in coffee subsequent to divergence from Arabidopsis, or rather a loss of the element in the Arabidopsis lineage. Second, the fact that this retrotransposon gag protein element was discovered in an EST-database indicates that this particular retroelement is being transcribed in the coffee genome, and hence may represent an active retrotransposon.
Another gene family for which coffee has significantly more members than Arabidopsis, encodes proteins annotated in Arabidopsis as acid endochitinases and photoassimilate-responsive proteins (Table 6). As noted earlier, chitinases are associated with fungal resistance and are among the most highly expressed genes in coffee. The fact that chitinases are both highly expressed and represented by an expanded gene family in coffee may reflect a greater need for fungal resistance engendered both by the perennial nature of coffee and the fact that it is a tropical species for which a multiplicity of fungal pathogens is common. The reasons for the putative expansions of the other gene families listed in Table 6 remain for future studies to determine.
Coffee-unique gene families
Table 7 lists the top gene families (based on copy number), which occur in coffee, but not in Arabidopsis. Of the 15 gene families listed, four are of unknown function. For those that could be functionally annotated, five (45%) have putative functions related to disease resistance, such as TIR-NBS-LRR disease resistance proteins, LRR proteins and thaumatin pathogenesis-related proteins (Table 7). These finding are consistent with rapid evolution of genes/gene families related to disease resistance, likely driven by selection pressure from continuously changing pathogens and/or pathogens unique to the particular environments of a species (Meyers 1998; Michelmore and Meyers 1998). Also included in this list of coffee-unique gene families are two, which encode retrotransposon gag-proteins (Table 7).
Comparison of the coffee gene repertoire with that of Arabidopsis and Solanaceae species
Each coffee unigene was screened against the Arabidopsis proteome using BLASTX, which compares all six translated frames of the query with the target. In total, 21% of the coffee unigenes had no clear match in Arabidopsis (match score 0–30) (Fig. 6). These coffee genes, which lack an Arabidopsis counterpart, may have evolved rapidly since coffee and Arabidopsis diverged from their last common ancestor, and hence their counterparts in each genome are no longer recognizable by standard string searches like BLAST. To investigate this topic further, the 2,853 unigenes with no clear match in Arabidopsis (match score<30, see Fig. 6) were screened against the Solanaceae EST-derived unigene sets using TBLASTX which compares all six translated frames of both query (coffee unigenes) and target (Solanaceae unigenes, see Materials and methods for details) sequences. The family Solanaceae is much more closely related to coffee than is Arabidopsis (Fig. 1), and hence coffee genes without a match in Arabidopsis may well have recognizable orthologs in the Solanaceae. The results show that a large portion (90%) of the coffee genes that lack a match in Arabidopsis do have a match in Solanaceae at the same stringency criteria (match score>30). For many of these cases, however, the coffee-Solanaceae matches are still quite diverged (low match scores) suggesting that the proteins encoded by these genes have been evolving at a rapid rate in dicots.
Fig. 6Histogram showing match scores for each coffee unigene as compared with its best match in the Arabidopsis proteome
There were, however, some exceptions to this trend represented by coffee genes having no match in Arabidopsis, yet a strong match in Solanaceae. These latter cases may represent genes that have been under differential selection pressure since the time that the Rosids (which includes Arabidopsis) diverged from the last common ancestor with the Asterids (which includes Solanaceae and coffee) (Fig. 1). Alternatively, these genes may have been lost in the lineage leading to Arabidopsis, but retained in the Asterids. In either case, such genes may have functional roles that are, in part, responsible for the morphological, developmental and chemical characteristics that differentiate Arabidopsis from Rosid species (e.g. coffee, Solanaceae). In an effort to shed more light on this issue, the 20 coffee unigenes having the highest match scores with Solanaceae, but lacking a counterpart in Arabidopsis were parsed and annotated (Table 8). Seven of these coffee genes have no match either in GenBank NR or dbest hence their function is unknown. Another unigene (Unigene 125230, the 2s seed storage protein, see above discussion) had weak match to ESTs of sesame, a species also belonging to the Asterid I clade (Fig. 1). Thus, these eight genes (40%) appear to be specific to the tomato/coffee/sesame Asterid I clade (Fig. 1, Table 8). Elucidating their function may shed light on the molecular processes, which diversified early in the evolution of dicot species, especially those species belonging to the Asterid clade of dicots.
Table 8Coffee genes not found in Arabidopsis, but with conserved counterparts in tomato or other Solanaceous speciesCoffee unigeneSolanaceae EST-derived unigene matchScoreGenBank (non-redundant and dbest) best matchScoreAnnotation124978240871 tomato454Unknown function121324235756 tomato429gblCB686389.1 [Brassica napus]44Unknown function131820213100 tomato426gil50252229.1 [Oryza sativa]73Unknown function121542240321 tomato416refiNP_922676.1 [Oryza sativa]75Unknown function131934219759 tomato377embiCAE05735.1 [Oryza sativa]297TFIIH basal transcription factor p52 subunit121140236347 tomato320Unknown function131445225435 tomato320Unknown function125230243065 tomato238gil13183175 [Seasame indicum]452S albumin131030246364 potato213refINP_524404.1 [Drosophila melanogaster]110Phospyhatidyl inositol transfer protein120120237254 tomato202gbICF349465.1 [Rose]52Unknown function126635237314 tomato185Unknown function126575237314 tomato182Unknown function130675209387 petunia177gbICK093976.1 [Populus tremula]438Unknown function128020237314 tomato167Unknown function123615249253 potato163gbIAAO73272.1 [Oryza sativa]140Unknown function126432240551 tomato163gil34878866 [Rattus norvegicus]56Phosphatidylinositolglycan class N124384197378 pepper156gblCA815435.1 [Vitis vinifera]1,009Unknown function122126239632 tomato153Unknown function131601232010 tomato145gbICK229938.1 [Macaca mulatta]7440S Ribosomal protein S21119644237150 tomato143refINP_921250.1 [Oryza sativa]70HelicaseThe GenBanks Best match exclude those from Solanaceae, Coffea and Hedyotis (both members of the Rubiaceae family). Solanaceae EST-derived
Surprisingly, 8 (40%) of the 20 coffee genes having no match in Arabidopsis did have matches in species phylogenetically distant from both coffee and Arabidopsis, including two to non-plant species (Drosophila and rat) (Table 8). Five of these matches were to rice genes, which is a monocot and highly divergent from coffee, Arabidopsis and other dicot species (Table 8). The fact that all of these species diverged from Arabidopsis and coffee long before the latter diverged from each other suggests that these genes may have been present in the last common ancestor of Arabidopsis and coffee/Solanaceae, but subsequently lost in the Arabidopsis lineage.
Coffee genes share greater similarity to genes in tomato/Solanaceae than to Arabidopsis
As discussed earlier, coffee is much more closely related to the Solanaceae than to Arabidopsis (Fig. 1). Hence, Solanaceae species may be better models for coffee genomics than Arabidopsis. The results for fast-evolving genes, presented above, are consistent with this prediction. To further investigate this assertion, the degree to which each coffee unigene matched Arabidopsis versus Solanaceae was investigated. In doing this analysis, one has to keep in mind that the entire gene repertoire of Arabidopsis is known, whereas the EST-derived unigene sets for Solanaceae do not represent the entire gene repertoire of these species. We estimate that the combined EST-derived unigene sets of Solanaceae species represent as much as three-quarters of the Solanaceae gene content (Hoeven et al. 2002). Moreover, Arabidopsis genes are of full length, while Solanaceae EST-derived unigenes are not necessarily of full length.
To mitigate against these factors, each coffee unigene was first screened, via TBLASTX, against the Solanaceae EST-derived unigene databases. Only those coffee unigenes matching a Solanaceae unigene with a match score greater than 100 were compared with the Arabidopsis proteome. Moreover, the match to Arabidopsis was confined to only the portion of the coffee unigene matching a Solanaceae EST-derived unigene. The ratio of the Arabidopsis match score divided by the Solanaceae match score was used as a measure of the degree to which the Solanaceae homolog shows greater similarity to the coffee unigene than to the closest Arabidopsis homolog. The results of this analysis are shown in Fig. 7. The ratio of the Arabidopsis/Solanaceae match score averaged 0.75, indicating a significantly better match in most cases with Solanaceae versus Arabidopsis. Moreover, in more than 95% of the cases, the ratio was less than one, indicating that, in most instances, coffee contains genes significantly more homologous to Solanaceae than to Arabidopsis. For those cases in which the ratio was greater than 1 (5%), we cannot rule out the possibility that the true ortholog was missing in the Solanaceae EST-derived unigene sets, hence the comparison was between paralogs. These results, combined with the results on fast evolving genes presented earlier, provide clear evidence that the Solanaceae provide a much better model than Arabidopsis for genomic and biological studies in coffee. This is especially relevant as sequencing of the tomato genome is currently underway (http://www.sgn.cornell.edu/help/about/tomato_sequencing.html).
Fig. 7Ratio of highest Arabidopsis match score to highest Solanaceae match score for individual coffee unigene. The analysis restricted to coffee unigenes with a Solanaceae match score >100
Conclusions
Herein, we describe the development and analysis of a large EST database for coffee. The resulting 47,000 ESTs correspond to 13,175 unique genes (unigenes), a large portion of which are expressed during seed development—a stage important to coffee as a crop and one for which our understanding of molecular development is still rudimentary. To our knowledge, this is the largest public database for seed-derived ESTs. Hence, this EST database represents a new public resource, which can facilitate a better understanding of seed development, as well as genomic, molecular and breeding research in coffee. By comparisons with Arabidopsis and Solanaceous species, we have identified the two major seed storage proteins of coffee (2S and 11S) and demonstrated that these proteins are expressed at different times during seed development. Through in silico gene expression analysis, we have identified a number of highly expressed genes that show high specificity for different stages of seed development as well as for the pericarp tissue that surrounds the seeds. Many of these highly expressed genes are unique to coffee and/or the Asterid clade of higher plants. While the functions of most of these highly expressed, tissue/stage specific genes remain to be determined, the fact that they have been identified points the way to promoters, which can potentially be used to drive gene expression in specific stages/tissues of the coffee plant. Many of these genes are specific to defined periods of seed and/or pericarp development—both critically important for insect/pathogen resistance and in determining the quality of the coffee bean with respect to commercial coffee products.
Coffee, as a member of the family Rubiaceae, is distantly related to the model species Arabidopsis. A computational comparison of the coffee EST-derived unigene set with the sequence databases for Arabidopsis and Solanaceous species (e.g. tomato, pepper), indicate that the latter are much better genomic models for coffee than is Arabidopsis. These results are consistent with the fact that coffee and solanaceous species share very similar chromosome architecture and are closely related, both belonging to the Asterid I clade of dicot plant family. Moreover, the ability to identify orthologous genes between coffee and tomato opens the door to eventually developing detailed comparative maps for these two species and to the sharing of genomic and biological tools/discoveries—an outcome that should expedite research in both taxa.

solanaceae
coffea canephora
seed development
rubiaceae
comparative genomics


The CD38/CD157 mammalian gene family: An evolutionary paradigm for other leukocyte surface enzymes

Human CD38 is the mammalian prototype of a family of phylogenetically conserved proteins which share structural similarities and enzymatic activities involved in the production of an intracellular second messenger with calcium mobilizing effects. Engagement of CD38 by agonistic monoclonal antibodies and the CD31 ligand initiates a cytoplasmic signaling cascade involving tyrosine phosphorylation of the proto-oncogene c-cbl and of the extracellular regulated kinase 1 of 2 complex. Further requirements for signal transduction include a privileged localization within the cholesterol-rich areas of the plasma membrane and physical association with specialized surface receptors. CD38-mediated signals are crucial in heterotypic cell adhesion and migration as well as in the activation of proliferation/survival programs by normal and neoplastic cells. Here we review the most recent literature on this complex topic and attempt to formulate a single model reconciling the enzymatic and receptorial activities of CD38.

calcium mobilization
cell surface enzymes
cell surface receptors
intracellular signaling


Angiographic CT with intravenous administration of contrast medium is a noninvasive option for follow-up after intracranial stenting

Intracranial angioplasty and stenting (ICAS) is a therapeutic option in symptomatic intracranial atherosclerotic disease. Adequate follow-up examination is necessary to exclude in-stent restenosis. Conventional intraarterial digital subtraction angiography (ia-DSA) is the current gold standard, but it is an invasive technique and carries the risk of neurological complications. Angiographic CT (ACT) is a new technique that provides a volume dataset of the highest spatial resolution and high contrast resolution derived from a rotational acquisition of a c-arm-mounted flat-panel detector. The feasibility of ACT with intravenous administration of contrast medium (iv-ACT) for follow-up after ICAS is demonstrated. In two patients iv-ACT was performed as a follow-up examination 12 months after ICAS. High-resolution volume data from the rotational acquisitions were processed to provide delineation of the stent lumen as well as imaging of the brain parenchyma and vessels. In both patients the patency of the stent lumen was assessed successfully. In addition, all other brain vessels were displayed in a manner similar to their appearance on CT angiograms. The brain parenchyma was also adequately imaged in a manner similar to its appearance on CT images. We demonstrated the feasibility and diagnostic value of iv-ACT for follow-up imaging after ICAS. This new application has the potential to become the imaging method of choice after ICAS since it not only enables visualization of the patency of the stent lumen but also is minimally invasive and provides additional information about all brain arteries and the brain parenchyma.
Introduction
In symptomatic intracranial atherosclerotic disease refractive to medical treatment intracranial angioplasty and stenting (ICAS) is a therapeutic option increasingly used in specialized stroke centers. However, restenosis rates ranging from 8% to 30% have been reported, underlining the necessity for regular follow-up examinations to exclude significant restenosis [1–4]. CT angiography (CTA) and MR angiography (MRA) are known to suffer from considerable artifacts that often prevent assessment of the stent lumen. Therefore, current follow-up techniques include transcranial Doppler sonography and conventional intraarterial digital subtraction angiography (ia-DSA). However, diagnostic ia-DSA is an invasive technique with a rate of examination-related neurological complications ranging from 0.5% to 0.8% [5, 6]. In addition, ia-DSA is not offered on an outpatient basis at every medical center so patients have to be admitted for at least one night, significantly increasing the treatment costs. Furthermore, patient compliance with follow-up examinations may be a problem because the ia-DSA procedure is not very comfortable.
The new angiographic systems equipped with flat-panel detectors which allow rotational acquisition of volume data provide the possibility of CT-like low-contrast imaging [7]. The administration of even highly diluted contrast medium intraarterially gives good delineation of the vessels indicating that intravenous administration of contrast medium could be sufficient. We sought to combine this property with the possibility of nearly artifact-free imaging of small-vessel stents described previously [8].
Methods
DSA and angiographic CT (ACT) examinations were performed on an AxiomArtis dBA biplane angiography system equipped with flat panel detectors (Siemens Medical Solutions, Forchheim, Germany). The acquisition of rotational datasets was performed with the following parameters (20s-1k protocol): 20 s rotation, 538 projections, 220° total angle, no zoom (detector size 30×40 cm), CTDIweighted approximately 22 mGy (manufacturer’s information). Postprocessing of the image data to a volume dataset (ACT) was performed on a Leonardo workstation (DynaCT, InSpace 3D software, Siemens). The software includes the application of system-specific filter algorithms in order to correct for beam hardening, radiation scatter, truncated projections and ring artifacts. Postprocessing resulted in volume datasets each defined by a batch of about 400 slices in a 512×512 matrix. Voxel size was about 0.1×0.1×0.1 mm3. The ACT datasets were further processed to multiplanar reconstructed (MPR) slices with the thin-slice and thick-slice maximum intensity projection (MIP) technique parallel and perpendicular to the stent-supplied section of the vessel, respectively. The contrast medium used in the follow-up ACT examination was Imeron 350 (Bracco ALTANA Pharma, Konstanz, Germany). The contrast medium was injected to a volume of 100 ml into a cubital vein at a flow rate of 5 ml/s, and the start delay for rotational acquisition was 20 s. The patient was asked to close his or her eyes and to breathe calmly during the examination.
CTA was performed on a 16-slice CT scanner (Aquilion, Toshiba Medical Systems, Tokyo, Japan) with 0.5-mm slice collimation, intravenous injection of 60 ml Imeron 350 (Bracco ALTANA Pharma), and a CTDIvol of approximately 50 mGy [9]. Postprocessing was performed on a Vitrea II workstation (Vital Systems, Minnetonka, Minn.).
Case illustrations
Case 1
A 57-year old man suffering from brainstem infarction and recurring transient ischemic attacks of the vertebrobasilar circulation was admitted to our hospital. Transcranial ultrasonography and MRA revealed a high-grade stenosis of the basilar artery. The symptoms occurred despite double platelet inhibition (aspirin and clopidogrel) and ICAS was chosen as therapy. The procedure was performed under general anesthesia. A balloon-mounted Pharos (3.5/8 mm) stent (Micrus Endovascular, Renens, Switzerland) was placed and balloon-inflated to a diameter of 3.2 mm thereby reconstructing a diameter corresponding to about 90% of the normal vessel diameter (Fig. 1a,b). The successful deployment of the stent was documented by native ACT (Fig. 1c,d). No complications occurred after the intervention and the patient was discharged 3 days later.
Fig. 1Patient 1. a, b DSA with selective injection of contrast medium into the right vertebral artery shows the eccentric high-grade stenosis of the basilar artery (a). After stent placement (b) the lumen of the basilar artery is well reconstructed and considerable inflow from the contralateral vertebral artery can be seen. c, d Reformatted images in the sagittal plane from an ACT scan directly following the intervention reveal regular deployment of the stent (c MIP, 0.5-mm slice thickness; d MIP, 5-mm slice thickness)
An outpatient follow-up CTA was performed 6 months after the procedure. The stent lumen was not adequately visualized on thin-slice MPR (Fig. 2a). Therefore, admission for follow-up DSA was organized for 2 months later. The patient refused ia-DSA, so ACT with intravenous administration of contrast medium (iv-ACT) was offered as an alternative. Written informed consent for the examination as a novel application of a rotational acquisition was obtained. The examination was successful. Corresponding to the good low-contrast resolution of ACT described previously, the reconstructed images revealed full patency of the stent lumen (Fig. 2b,c) [10]. This result was highly correlated with the flow-rate measured by transcranial Doppler sonography that had remained normal from directly after the intervention until the 8-month follow-up.
Fig. 2Patient 1. a CTA image obtained at the 6-month follow-up is not of sufficient quality to reveal the stent lumen, and in particular the proximal and distal markers produce substantial artifacts (MIP, 1-mm slice thickness). b, c iv-ACT images obtained at the 8-month follow-up show an unchanged configuration of the stent in comparison to the examination performed directly after the intervention (see Fig. 1c,d). The contrast filling is more faint than on the CTA image but the patency of the stent lumen is visible much more clearly on the sagittal cross sections, and the artifact load is acceptable (b MIP, 0.5-mm slice thickness; c MIP, 5-mm slice thickness)
Case 2
An 82-year-old man was admitted suffering from progressive left-sided hemiparesis and impaired consciousness. CTA revealed a high-grade stenosis of the right middle cerebral artery (MCA) and a second middle-grade stenosis of the basilar artery (data not shown). CT-perfusion (CTP) showed a prolonged mean transit time in the whole right MCA territory but only small infarcted areas level with the basal ganglia, represented by lowered relative cerebral blood volume. Emergency ICAS was performed under general anesthesia. A balloon-mounted Driver (2.5/8 mm) stent (Medtronic, Minneapolis, Minn.) was successfully placed in the stenosis of the right M1 segment leading to a good morphological result and immediate restoration of normal blood flow (Fig. 3a,b). Proper stent deployment was documented by native ACT (Fig. 3c,d).
Fig. 3Patient 2. a, b DSA images with selective injection into the right internal carotid artery reveals a high-grade stenosis of the right middle cerebral artery (MCA) with reduced distal blood flow (a). After stent placement the blood flow is normalized (b). c, d Native ACT reformatted images in paracoronal orientation reveal regular deployment of the stent (c MIP, 0.5-mm slice thickness; d MIP, 5-mm slice thickness)
The patient was admitted for follow-up DSA 12 months after the procedure. Transcranial Doppler sonography showed a normal flow velocity in the right MCA. This patient also refused ia-DSA. Written informed consent was obtained in advance to perform iv-ACT and to omit ia-DSA if the iv-ACT was of diagnostic quality. The procedure was performed successfully and the reconstructed images revealed full patency of the stent lumen (Fig. 4a,b). All other intracranial arteries were visible at the highest resolution, revealing an unchanged configuration of the medium-grade stenosis of the basilar artery (Fig. 4c,d). The reconstructed brain parenchyma images showed already known residual infarction (data not shown).
Fig. 4Patient 2. a, b Iv-ACT images obtained 12 months after the intervention reveal an unchanged stent configuration and full patency of the stent lumen (a MIP, 0.5-mm slice thickness; b MIP, 10-mm slice thickness). Even small perforating arteries originating through the stent struts can be seen (barrow). c A transverse thick-slice (30 mm) MIP overview image gives a good impression of the insular MCA branches of both sides. d A second thick-slice (15 mm) MIP image oriented parallel to the basilar artery reveals a medium-grade stenosis (arrow) and an anatomic variant of the origin of the superior cerebellar artery
Discussion
In patients suffering from intracranial atherosclerosis, ICAS is a promising new therapeutic option [1, 3, 11]. To identify an in-stent restenosis a reliable imaging technique is needed. Here, DSA is the current gold standard, but it carries a 0.5% to 0.8% risk of permanent neurological impairment [5, 6]. Additionally, not every center is able to perform ia-DSA on an outpatient basis. The risk may be less with ia-ACT if the injection is performed in the aortic arch. However, this is still an invasive procedure.
In the study reported here we demonstrated that iv-ACT can reliably depict the lumen of small-vessel stents with high spatial resolution. As the low contrast resolution of ACT is comparable to that of conventional CT, a relatively low concentration of intravascular contrast medium is needed. All intracranial vessels can be viewed simultaneously in a high-quality image. CT-like images of the brain parenchyma are provided as well [10]. It therefore represents a new radiological imaging technique between CTA and ia-DSA.
Even though this new method needs to be evaluated in a larger number of patients, it possibly has many advantages for patients and physicians as a new option in the imaging of cerebrovascular disease: Examinations can be performed on an outpatient basis and the risk of neurological complications is lowered practically to zero. The examination can be performed quickly, in a time similar to that required for a CTA examination. The radiation exposure for the patient from a rotational acquisition is lower than that from a CTA examination (manufacturer’s information) and has been shown to be considerably lower than that from a single biplane DSA series [12].
Possible drawbacks of the iv-ACT technique first include movement artifacts. The patient has to lie nearly motionless for about 20 seconds, which requires a high level of compliance and is not always feasible in patients suffering from cerebrovascular disease.
The first stent we examined is a relatively new balloon-mounted stent designed for intracranial use (Pharos, Micrus Endovascular, Renens, Switzerland). All dedicated intracranial stents (Neuroform and Wingspan, Boston Scientific, Natick, Mass.; LEO, BALT, Montmorency, France; and Enterprise, Cordis, Miami Lakes, Fl.) have a similar strut size of about 0.06 mm [13]. In contrast, common coronary stents have thicker struts (e.g. Driver, Medtronic, Minn.: 0.09 mm) [14]. Our second patient demonstrates that the lumen of these stents can be visualized as well. This issue is not unimportant because until recently dedicated intracranial stents were not available. Therefore, the majority of stents implanted into intracranial vessels are still coronary stents.
Iv-ACT is a promising new technique for minimally invasive follow-up after intracranial stenting. Other possible applications can be envisaged, such as diagnostic and follow-up imaging before and after endovascular treatment of intracranial aneurysms or arteriovenous malformations.

intracranial stenting
flat-panel
cerebrovascular disease
rotational angiography
angiographic computed tomography


Spectrofluorimetric Determination of Oxamniquine in Dosage Forms and Spiked Human Plasma through Derivatization with 1-dimethylaminonaphthalene-5-sulphonyl chloride

A sensitive, simple and selective spectrofluorimetric method was developed for the determination of oxamniquine (OXM) in pharmaceutical formulations and biological fluids. The method is based on the reaction between the drug and 1-dimethylaminonaphthalene-5-sulphonyl chloride (dansyl chloride) in presence of 0.5 M sodium carbonate (pH 10) to yield a highly fluorescent derivative that is measured at 445 nm after excitation at 335 nm. The different experimental parameters affecting the development and stability of the reaction product were carefully studied and optimized. The fluorescence concentration plot was rectilinear over the range of 0.02–0.2 μg ml−1 with a lower detection limit (LOD) of 0.007 μg ml−1 and limit of quantitation (LOQ) of 0.02 μg ml−1. The proposed method was successfully applied to the analysis of commercial capsules. The results obtained were in good agreement with those obtained using the official spectrophotometric method. Furthermore, the method was applied for the determination of oxamniquine in spiked human plasma, the mean % recovery (n = 4) is 97.77 ± 1.19. A proposal of the reaction pathway was presented.
Introduction
Oxamniquine (OXM), 1,2,3,4-tetrahydro-2[(isopropylamino methyl]-7-nitro-6-quyinoline methanol is an antischistosomal agent that is indicated for the treatment of schistosoma mansoni (intestinal schistosomiasis) infection (Fig. 1). It has been shown to inhibit DNA, RNA and protein synthesis in schistosomes. The oral bioavailability of oxamniquine is good and effective plasma levels are achieved in 1–1.5 h [1].
Fig. 1Structural formula of oxamniquine
Oxamniquine is the subject of a monograph in the USP (XXIII) [2] whereby a spectrophotometric method is recommended for its determination, whether in its pure form or in capsules. Several methods have been published for the determination of OXM, either per se or in pharmaceutical preparations and biological fluids. These methods include: spectrophotometry [3–5], non-aqueous titration [6], gas chromatography [7], HPLC [8–13], capillary electrophoresis [11], polarography [14], flow injection analysis [15], cyclic voltammetry [16] and fluorimetry [17].
All these methods are either insufficiently sensitive or tedious and require highly sophisticated and dedicated instrumentation [8–13]. This led us to study the reaction of oxamniquine with dansyl chloride to develop simple and sensitive spectrofluorimetric method for its determination in pharmaceutical preparations and biological fluids. Dansyl chloride is a useful derivatizing agent for primary amines, secondary amines, imidazoles and phenols, etc.. Several pharmaceutical compounds have been determined through this approach [18–24].
The reported spectrofluorimetric method for oxamniquine [17] is tedious and time consuming. It involves the use of 2-cyanoacetamide, a hazard reagent.
Experimental
Apparatus
The spectrofluorimetric measurements were recorded using ARF-1501 Shimadzu Spectrofluorometer, equipped with Xenon arc lamp.
Materials and reagents
All reagents and solvents were of analytical reagent grade
Oxamniquine pure sample was kindly provided by Pfizer (Sandwich, UK).Capsules containing 250 mg of Oxamniquine each (Vansil capsules) were obtained from commercial sources in the local market.1-Dimethyl aminonaphthalene-5-sulphonyl chloride (dansyl chloride), purchased from Sigma (St. Louis, USA). A stock solution containing 0.1% of dansyl chloride was freshly prepared in acetone and was further diluted with the same solvent to obtain 0.001% solution.Sodium carbonate (BDH, UK) 0.5 M aqueous solution (pH 10).Sodium hydroxide (BDH, UK) 1 M aqueous solution.Isobutylmethyl ketone (IBMK) (Merck, Darmstadt, Germany).Plasma was kindly provided by Mansoura University Hospital, and kept frozen until assay after gentle thawing.Methanol and diethyl ether (Merck, Darmstadt, Germany).
Standard solutions
A stock solution was prepared by dissolving 20.0 mg of OXM in 20 ml of acetone and 80 ml of 0.5 M sodium carbonate solution. This solution was further diluted with the same solvent mixture as appropriate. The standard solutions were stable for seven days when kept in the refrigerator.
General procedure
Aliquots of OXM standard solution were transferred into a series of 10 ml volumetric flasks. 0.7 ± 0.1 ml of 0.001% of dansyl chloride reagent was added, followed by 0.4 ml of acetone and mixed well. The reaction mixture was left for 30 min, and then completed to the mark with IBMK. the reaction mixture was allowed to stand for 10 min. The fluorescence intensity of the reaction product was measured at 445 nm after excitation at 335 nm. Blank experiment was carried out simultaneously. The corrected fluorescence intensity was plotted vs the final drug concentration (μg ml−1) to get the calibration graph. Alternatively, the corresponding regression equation was derived.
Applications
Procedure for commercial capsules
The content of ten capsules were emptied, and mixed well. A weighed quantity of the powder equivalent to 5.0 mg OXM was transferred into a small conical flask, and extracted with 2 × 10 ml of methanol. The extract was filtered into 25 ml volumetric flask. The conical flask was washed with several milliliters of methanol. The washing was passed into the same volumetric flask, and the combined extract was evaporated to dryness on a boiling water bath. The residue was dissolved and diluted to volume with a mixture of 5 ml of acetone and 20 ml of 0.5 M of sodium carbonate solution. Aliquots covering the working concentration range (cited in Table 1) was transferred into 10 ml volumetric flasks. The “General procedure” was then applied. The nominal content of the capsules was determined either from the calibration curve or using the corresponding regression equation.
Table 1Performance data of the proposed methodParameterProposed methodConcentration range (μg ml−1).0.02–0.2Minimum detection limit, LOD (μg ml−1).0.007Limit of Quantification , LOQ(μg ml−1).0.02Correlation coefficient ( r).0.9999Slope3,473.114Intercept0.574Sy/x1.273Sa7.733Sb8.965% Error0.28Sy/x = standard deviation of the residualsSa = standard deviation of the intercept of regression lineSb = standard deviation of the slope of regression line% Error = RSD%/√ n
Procedure for spiked human plasma
A stock solution containing 20 μg ml−1 of OXM was prepared. Control samples of plasma was spiked with different quantities of OXM to give a final drug concentration cited in Table 5. One molar NaOH (0.8 ml) was added to 1.0 ml of the spiked plasma and shaken genteelly. The solution was vortexed with 3 × 5 ml of diethylether for 2 min, then centrifuged at 2,500 rpm for 5 min. The resulting supernatant was evaporated to dryness under nitrogen at ambient temperature. The residue was dissolved and diluted to volume with a mixture of 5 ml of acetone and 20 ml of 0.5 M of sodium carbonate solution. Aliquots covering the working concentration range was transferred into 10 ml volumetric flasks. The recommended procedures were then applied. The nominal content of the drug was determined using the corresponding regression equation.
Results and discussion
Dansyl chloride was first introduced for the determination of some primary, secondary amines, imidazoles and phenols [18–20].
In recent reports. DNS was further used as a fluorogenic reagent for the determination of some pharmaceutical compounds [21–24].
In the present study, OXM was found to react with Dansyl chloride at pH 10.0 forming a highly yellow fluorescent derivative with λ maximum emission at 445 nm after excitation at 335 nm (Fig. 2).
Fig. 2Fluorescence spectra of A and B excitation and emission spectra of blank. Fluorescence spectra of A′ and B′ excitation and emission spectra of oxamniquine (0.2 μg ml−1) with dansyl chloride at pH 10.0
Optimization of experimental parameters
The spectrofluorimetric properties of the colored product as well as the different experimental parameters affecting the development of the reaction product and its stability were carefully studied and optimized. Such factors were changed individually while the others were kept constant. The factors include pH, concentration of the reagent, type of buffer, temperature, reaction time and dilution time.
Effect of pH
The influence of pH on the fluorescence intensity of the reaction product was studied. Maximum fluorescence intensity was obtained upon using mixture of acetone and 0.5 M sodium carbonate solution. The pH of the reaction mixture was found to be 10.0. pH 10 was found to be the optimum pH for dansylation because labeling of most amino acids, amines, imidazoles and phenols has been found to be optimal at pH 9.5–10.5 [25]. The rate of dansylation process was found to increase with increasing the pH value this is due to an increase in the rate of hydrolysis of dansyl chloride into dansyl hydroxide [25]. The latter shows strong fluorescence and hence interferes seriously in the determination. However, under the proposed chosen conditions and wavelengths used, there was no interference arising from any dansyl hydroxide formed, as indicated by the low fluorescence intensity of the reagent.
Effect of concentration of dansyl chloride
The influence of the concentration of dansyl chloride was studied using different volumes of 0.001% of the reagent solution. It was found that, the reaction of dansyl chloride with OXM started upon using 0.1 ml of the reagent in the presence of sodium carbonate (pH 10.0). Increasing the volume of the reagent, produces a proportional increase in the fluorescence intensity of the reaction product up to 0.6 ml and remains constant up to 0.8 ml. Therefore, 0.7 ± 0.1 ml of 0.001% of dansyl chloride solution was chosen as the optimal volume of the reagent (Fig. 3).
Fig. 3Effect of volume of dansyl chloride (0.001%) on the fluorescence intensity of the reaction product of oxamniquine (0.2 μg m−1) at pH 10.0
Effect of temperature
Increasing the reaction temperature higher than the room temperature would result in a subsequent decrease in the fluorescence intensity of the reaction product.
Effect of reaction time
Different time intervals were tested to ascertain the time after which the solution attains its highest fluorescence intensity. It was found that after 30 min, the reaction product reaches the highest fluorescence intensity (Fig. 4) and remains stable at room temperature for 20 min.
Fig. 4Effect of reaction time of on the fluorescence intensity of the reaction product of oxamniquine (0.2 μg ml−1) with dansyl chloride
Effect of diluting solvent
Different solvents were tried to dilute the reaction mixture through out the study. It was observed that isobutyl methyl ketone gave the highest fluorescence intensity. Dilution with 0.5 M sodium carbonate solution, water, acetone–water produced almost very week fluorescence and did not reduce the blank fluorescence intensity. While upon using isobutyl methyl ketone, the fluorescence intensity attained its highest value, this was attributed to the low fluorescence value of the reagent.
Effect of dilution time
Dilution times were tested to ascertain the time after which the solution attains its highest fluorescence. It was found that dilution with IBMK after 10 min, the reaction product reaches its highest fluorescence intensity (Fig. 5).
Fig. 5Effect of dilution time on the fluorescence intensity of the reaction product of oxamniquine (0.2 μg ml−1) with dansyl chloride
Analytical performance
Validation of the proposed methods The validity of the method was tested regarding; linearity, specificity, accuracy, repeatability and precision according to ICH Q2B recommendations [26].
Linearity
By using the above procedure, linear regression equation was obtained. The regression plots showed that there was a linear dependence of the fluorescence intensity on the concentration of the drug over the ranges cited in Table 1. Linear regression analysis of the data gave the following equation:
where F is the fluorescence intensity, C is the concentration of the drug in μg ml−1 and r is the correlation coefficient.
The limit of quantification (LOQ) was determined by establishing the lowest concentration that can be measured according to ICH Q2B [26]. The results are shown in Table 1. The limits of detection (LOD) were determined by establishing the minimum level at which the analyte can be reliably detected, and the results are also abridged in Table 1.
LOQ and LOD were calculated according to the following equation [26]:
Where σ: is the standard deviation of the intercept of regression line. S: is the slope of the calibration curve.
The proposed methods were evaluated for the accuracy as percent relative error (% Er) and the precision as percent relative standard deviation (% RSD) (Tables 1 and 2).
Table 2Application of the proposed and official methods to the determination of Oxamniquine in pure formParametersSpectrofluorimetric methodOfficial method [2]No. of experiments63Mean found (%)100.13100.09 ± SD0.680.87 % RSD0.680.87Variance0.4620.76Student’s t value0.081 (2.31)Variance ratio F test1.65 (5.41)Figures between parentheses are the tabulated t and F values respectively, at p = 0.05 [27]
Accuracy
To test the validity of the proposed method it was applied to the determination of authentic sample of OXM over the working concentration range. The results obtained were in good agreement with those obtained using official method. Using Student’s t test and variance ratio F test, [27] revealed no significant difference between the performance of the two methods regarding the accuracy and precision, respectively (Table 2).
The validity of the methods was proved by statistical evaluation of the regression lines, using the standard deviation of the residuals (Sy/x), the standard deviation of the intercept (Sa) and standard deviation of the slope (Sb). The results are abridged in Table 1. The small values of the figures point out to the low scattering of the points around the calibration line and high precision.
Precision
Repeatability
The repeatability was performed by applying the proposed methods for the determination of two concentrations of OXM in pure form on three successive times, and the results are listed in Table 3.
Intermediate precision
It was performed through repeated analysis of OXM in pure form, using the concentrations showed in Table 3 for a period of three successive days. The results are summarized in Table 3.
Table 3
Validation of the proposed method for the determination of oxamniquine in pure form
Sample concentration
% Recovery (repeatability)
% Recovery intermediate precision
0.08 μg ml−1
99.64
101.08
100.72
99.28
101.44
100.89
 X′
100.60
100.42
 ± SD
0.91
0.99
 %RSD
0.91
0.99
 % Error
0.53
0.57
0.2 μg ml−1
99.74
101.05
100.44
100.53
101.29
98.95
 X′
100.49
100.18
 ± SD
0.78
1.09
 %RSD
0.78
1.09
 % Error
0.45
0.63
Robustness of the method
The robustness of the method adopted is demonstrated by the constancy of the fluorescence intensity with the deliberated minor changes in the experimental parameters such as change in the volume of dansyl chloride (0.001%), 0.7 ± 0.1 ml, the change in reaction time 35 ± 5 min and the change in dilution time 15 ± 5 min. These minor changes that may take place during the experimental operation didn’t affect the fluorescence intensity of the reaction product.
Pharmaceutical applications
The proposed methods were then applied to the determination of OXM in its capsules. The methods were tested for linearity, specificity, accuracy, repeatability and precision according to ICH Q2B recommendations.
Specificity
The specificity of the method was investigated by observing any interference encountered from the common capsule excepients, such as talc, lactose, starch, avisil, gelatine, and magnesium stearate. These excepients did not interfere with the proposed method.
Accuracy
The results of the proposed methods were statistically compared with those obtained using the official method [2]. Statistical analysis [27] of the results, using Student’s t test and variance ratio F test revealed no significant difference between the performance of the proposed and reference methods regarding the accuracy and precision, respectively (Table 4).
Table 4Application of the proposed methods to the determination of oxamniquine in capsulesPreparations% RecoveryProposed methodOfficial method [2]Vansil capsules (250 mg oxamniquine/capsule)98.25101.0099.3899.8099.30 Mean found (%)± SD.99.55 ± 0.99100.56 ± 0.77 t value1.67 F value1.66The tabulated values of t and F are (2.37) and (6.59) respectively, at p = 0.05 [27]
Analysis of biological fluid
The high sensitivity of the proposed method allowed the determination of OXM in spiked human plasma. Oxamniquine is readily absorbed following oral ingestion, and a peak concentration in plasma occurs within about 3 h. The presence of food significantly delays absorption and limits the concentration achieved in plasma during the first several hours after administration. Urinary excretion is the major route of elimination in man [28]. Oxamniquine is given orally in a dose of 250 mg three times daily; this leads to a final blood level concentration of about 5 μg ml−1 i.e. higher than the upper limit of the working concentration range of the proposed method. The high sensitivity of the proposed method allowed the determination of OXM in spiked human plasma. The results are shown in Table 5.
Table 5Application of the proposed methods to the determination of oxamniquine in spiked human plasmaSampleAmount added (μg ml−1)Amount found (μg ml−1)% Recovery1-a-Plasma (inter-day precision)0.020.0195797.850.040.0385696.400.100.0975497.540.200.1985699.28 Mean97.77 ± SD1.191-b-Plasma (inter-day precision)0.200.191495.700.200.196998.450.200.194897.400.200.195597.750.200.197998.950.200.194597.25 Mean97.58 ± SD1.13Each result is the average of three separate determinations
The extraction procedure described by Woolhouse and Wood [7] was adopted here. The results are satisfactorily accurate and precise.
Precision The within-day precision was evaluated through replicate analysis of Plasma samples spiked with different concentrations of the drug. The percentage recoveries based on the average of four separate determinations were 97.77 ± 1.19, thus indicating the high precision of the method (Table 5).The inter-day precision was also evaluated through replicate analysis of plasma samples spiked with 0.2 μg ml−1 of drug on four successive days. The percentage recoveries based on the average of four separate determinations were 97.58 ± 1.13. The results are shown in Table 5.
Mechanism of the reaction
The stoichiometry of the reaction was studied adopting the limiting logarithmic method [29]. The fluorescence intensity of the reaction product was alternatively measured in the presence of excess of either dansyl chloride or OXM. A plot of log fluorescence versus log [dansyl chloride] and log [OXM] gave straight lines, the values of the slopes are 0.901 and 0.984 respectively (Fig. 6). Hence, it is concluded that, the molar reactivity of the reaction is 0.901/0.984, i.e. the reaction proceeds in the ratio of 1:1. Based on the observed molar reactivity of the reaction, and depending on the presence of secondary amino group and by analogy to similar reports dealing with the reaction of dansyl chloride with compounds containing secondary amino group, the reaction pathway proposed in Scheme 1 is presented.
Fig. 6Limiting logarithmic plots for the molar ratio. a Log F vs. Log [dansyl chloride]. b Log F vs. log [oxamniquine]Scheme 1Proposed reaction pathway between dansyl chloride and oxamniquine
Conclusion
The proposed method has the advantage of being simple, sensitive and suitable for routine analysis in quality control laboratory. Also, it is suitable for the determination of oxamniquine in spiked human plasma with minimum detection limit lower than the reported value. In addition, it could be applied to the determination of OXM in its pharmaceutical preparation.