Papers by Mohammed Motawia
International Journal of Biological Macromolecules, May 1, 2011
Starch phosphate is important in starch metabolism and in order to deduce its location and struct... more Starch phosphate is important in starch metabolism and in order to deduce its location and structural effects in clusters and building blocks of amylopectin, these were isolated from a normal potato (WT) and two starches with antisense suppressed glucan water dikinase (asGWD) activity and starch branching enzyme (asSBE) activity possessing suppressed and increased phosphate contents, respectively. Neutral N-chains and phosphorylated P-chains of the amylopectin macromolecules were similar in WT and asGWD, whereas asSBE possessed considerably longer P-chains. Cluster -limit dextrins were isolated by ␣-amylase treatment and successive -amylolysis. Cluster sizes were generally smaller in asSBE. The building block composition of neutral N-clusters were very similar in WT and asGWD, while asSBE was different, containing less blocks with degree of polymerization (DP) > 14. Phosphate content of the Pclusters of WT and asGWD was rather similar, while asSBE contained highly phosphorylated P-clusters with proportionally more P-chains and a low degree of branching. The average chain lengths of the Pclusters were, however, similar in all samples. Our data demonstrate only minor effect on the cluster structure in relation to phosphate deposition suggesting conserved reaction patterns of starch phosphorylation. Models are suggested to account for the principle structural and functional effects of starch phosphate esters.
ChemBioChem, Jun 24, 2005
A convergent block strategy for general use in efficient synthesis of complex α‐(1→4)‐ and α‐(1→6... more A convergent block strategy for general use in efficient synthesis of complex α‐(1→4)‐ and α‐(1→6)‐malto‐oligosaccharides is demonstrated with the first chemical synthesis of a malto‐oligosaccharide, the decasaccharide 6,6′′′′‐bis(α‐maltosyl)‐maltohexaose, with two branch points. Using this chemically defined branched oligosaccharide as a substrate, the cleavage pattern of seven different α‐amylases were investigated. α‐Amylases from human saliva, porcine pancreas, barley α‐amylase 2 and recombinant barley α‐amylase 1 all hydrolysed the decasaccharide selectively. This resulted in a branched hexasaccharide and a branched tetrasaccharide. α‐Amylases from Asperagillus oryzae, Bacillus licheniformis and Bacillus sp. cleaved the decasaccharide at two distinct sites, either producing two branched pentasaccharides, or a branched hexasaccharide and a branched tetrasaccharide. In addition, the enzymes were tested on the single‐branched octasaccharide 6‐α‐maltosyl‐maltohexaose, which was prepared from 6,6′′′′‐bis(α‐maltosyl)‐maltohexaose by treatment with malt limit dextrinase. A similar cleavage pattern to that found for the corresponding linear malto‐oligosaccharide substrate was observed.
Nature Communications, Aug 23, 2019
Synthetic biology efforts for the production of valuable chemicals are frequently hindered by the... more Synthetic biology efforts for the production of valuable chemicals are frequently hindered by the structure and regulation of the native metabolic pathways of the chassis. This is particularly evident in the case of monoterpenoid production in Saccharomyces cerevisiae, where the canonical terpene precursor geranyl diphosphate is tightly coupled to the biosynthesis of isoprenoid compounds essential for yeast viability. Here, we establish a synthetic orthogonal monoterpenoid pathway based on an alternative precursor, neryl diphosphate. We identify structural determinants of isomeric substrate selectivity in monoterpene synthases and engineer five different enzymes to accept the alternative substrate with improved efficiency and specificity. We combine the engineered enzymes with dynamic regulation of metabolic flux to harness the potential of the orthogonal substrate and improve the production of industrially-relevant monoterpenes by several-fold compared to the canonical pathway. This approach highlights the introduction of synthetic metabolism as an effective strategy for high-value compound production.
eLife, Mar 3, 2017
Despite vast diversity in metabolites and the matching substrate specificity of their transporter... more Despite vast diversity in metabolites and the matching substrate specificity of their transporters, little is known about how evolution of transporter substrate specificities is linked to emergence of substrates via evolution of biosynthetic pathways. Transporter specificity towards the recently evolved glucosinolates characteristic of Brassicales is shown to evolve prior to emergence of glucosinolate biosynthesis. Furthermore, we show that glucosinolate transporters belonging to the ubiquitous NRT1/PTR FAMILY (NPF) likely evolved from transporters of the ancestral cyanogenic glucosides found across more than 2500 species outside of the Brassicales. Biochemical characterization of orthologs along the phylogenetic lineage from cassava to A. thaliana, suggests that alterations in the electrogenicity of the transporters accompanied changes in substrate specificity. Linking the evolutionary path of transporter substrate specificities to that of the biosynthetic pathways, exemplify how transporter substrate specificities originate and evolve as new biosynthesis pathways emerge.
eLife, Mar 14, 2017
Forskolin is a unique structurally complex labdane-type diterpenoid used in the treatment of glau... more Forskolin is a unique structurally complex labdane-type diterpenoid used in the treatment of glaucoma and heart failure based on its activity as a cyclic AMP booster. Commercial production of forskolin relies exclusively on extraction from its only known natural source, the plant Coleus forskohlii, in which forskolin accumulates in the root cork. Here, we report the discovery of five cytochrome P450s and two acetyltransferases which catalyze a cascade of reactions converting the forskolin precursor 13R-manoyl oxide into forskolin and a diverse array of additional labdanetype diterpenoids. A minimal set of three P450s in combination with a single acetyl transferase was identified that catalyzes the conversion of 13R-manoyl oxide into forskolin as demonstrated by transient expression in Nicotiana benthamiana. The entire pathway for forskolin production from glucose encompassing expression of nine genes was stably integrated into Saccharomyces cerevisiae and afforded forskolin titers of 40 mg/L.
The Plant Cell, Oct 1, 2006
Two homologous plant-specific Arabidopsis thaliana genes, RGXT1 and RGXT2, belong to a new family... more Two homologous plant-specific Arabidopsis thaliana genes, RGXT1 and RGXT2, belong to a new family of glycosyltransferases (CAZy GT-family-77) and encode cell wall (1,3)-α-d-xylosyltransferases. The deduced amino acid sequences contain single transmembrane domains near the N terminus, indicative of a type II membrane protein structure. Soluble secreted forms of the corresponding proteins expressed in insect cells showed xylosyltransferase activity, transferring d-xylose from UDP-α-d-xylose to l-fucose. The disaccharide product was hydrolyzed by α-xylosidase, whereas no reaction was catalyzed by β-xylosidase. Furthermore, the regio- and stereochemistry of the methyl xylosyl-fucoside was determined by nuclear magnetic resonance to be an α-(1,3) linkage, demonstrating the isolated glycosyltransferases to be (1,3)-α-d-xylosyltransferases. This particular linkage is only known in rhamnogalacturonan-II, a complex polysaccharide essential to vascular plants, and is conserved across higher plant families. Rhamnogalacturonan-II isolated from both RGXT1 and RGXT2 T-DNA insertional mutants functioned as specific acceptor molecules in the xylosyltransferase assay. Expression of RGXT1- and RGXT2-enhanced green fluorescent protein constructs in Arabidopsis revealed that both fusion proteins were targeted to a Brefeldin A–sensitive compartment and also colocalized with the Golgi marker dye BODIPY TR ceramide, consistent with targeting to the Golgi apparatus. Taken together, these results suggest that RGXT1 and RGXT2 encode Golgi-localized (1,3)-α-d-xylosyltransferases involved in the biosynthesis of pectic rhamnogalacturonan-II.
bioRxiv (Cold Spring Harbor Laboratory), Dec 15, 2022
Carbohydrate Research, 2003
ABSTRACT
Plant Molecular Biology, Aug 7, 2015
Users may download and print one copy of any publication from the public portal for the purpose... more Users may download and print one copy of any publication from the public portal for the purpose of private study or research. You may not further distribute the material or use it for any profit-making activity or commercial gain You may freely distribute the URL identifying the publication in the public portal If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.
Journal of Natural Products, Mar 9, 2016
An effective method for the chemical synthesis of cyanogenic glucosides has been developed as dem... more An effective method for the chemical synthesis of cyanogenic glucosides has been developed as demonstrated by the synthesis of dhurrin, taxiphyllin, prunasin, sambunigrin, heterodendrin, and epiheterodendrin. O-Trimethylsilylated cyanohydrins were prepared and subjected directly to glucosylation using a fully acetylated glucopyranosyl fluoride donor with boron trifluoride−diethyl etherate as promoter to afford a chromatographically separable epimeric mixture of the corresponding acetylated cyanogenic glucosides. The isolated epimers were deprotected using a triflic acid/MeOH/ionexchange resin system without any epimerization of the cyanohydrin function. The method is stereocontrolled and provides an efficient approach to chemical synthesis of other naturally occurring cyanogenic glucosides including those with a more complex aglycone structure.
European journal of biochemistry, Dec 15, 2001
Enzymatic properties of barley a-amylase 1 (AMY1) are altered as a result of amino acid substitut... more Enzymatic properties of barley a-amylase 1 (AMY1) are altered as a result of amino acid substitutions at subsites 25/26 (Cys95 ! Ala/Thr) and þ1/þ 2 (Met298 ! Ala/ Asn/Ser) as well as in the double mutants, Cys95 ! Ala/ Met298 ! Ala/Asn/Ser. Cys95 ! Ala shows 176% activity towards insoluble Blue Starch compared to wild-type AMY1, k cat of 142 and 211% towards amylose DP17 and 2-chloro-4-nitrophenyl b-D-maltoheptaoside (Cl-PNPG 7), respectively, but fivefold to 20-fold higher K m. The Cys95 ! Thr-AMY1 AMY2 isozyme mimic exhibits the intermediary behaviour of Cys95 ! Ala and wild-type. Met298 ! Ala/Asn/Ser have slightly higher to slightly lower activity for starch and amylose, whereas k cat and k cat /K m for Cl-PNPG 7 are # 30% and # 10% of wild-type, respectively. The activity of Cys95 ! Ala/Met298 ! Ala/ Asn/Ser is 100-180% towards starch, and the k cat /K m is 15-30%, and 0.4-1.1% towards amylose and Cl-PNPG 7 , respectively, emphasizing the strong impact of the Cys95 ! Ala mutation on activity. The mutants therefore prefer the longer substrates and the specificity ratios of starch/Cl-PNPG 7 and amylose/Cl-PNPG 7 are 2.8-to 270-fold and 1.2-to 60-fold larger, respectively, than of wild-type. Bond cleavage analyses show that Cys95 and Met298 mutations weaken malto-oligosaccharide binding near subsites 25 and þ2, respectively. In the crystal structure Met298 CE and SD (i.e., the side chain methyl group and sulfur atom) are near C(6) and O(6) of the rings of the inhibitor acarbose at subsites þ1 and þ2, respectively, and Met298 mutants prefer amylose for glycogen, which is hydrolysed with a slightly lower activity than by wild-type. Met298 AMY1 mutants and wild-type release glucose from the nonreducing end of the main-chain of 6 000-maltotriosyl-maltohexaose thus covering subsites 2 1 to þ5, while productive binding of unbranched substrate involves subsites 2 3 to þ3.
Carbohydrate Research, Jul 1, 1999
A branched nonasaccharide 6 §-a-maltotriosyl-maltohexaose was synthesised in 40 steps from D-gluc... more A branched nonasaccharide 6 §-a-maltotriosyl-maltohexaose was synthesised in 40 steps from D-glucose and maltose. Phenyl O-(2,3,4,6-tetra-O-benzyl-aD -glucopyranosyl)-(1 4)-O-(2,3,6-triO -benzyl-aD -glucopyranosyl)-(1 4)-2,3-di-O-benzyl-1-thio-b-D-glucopyranoside and O-(2,3,4,6-tetra-O-benzyl-aD -glucopyranosyl)-(1 4)-O-(2,3,6-triO -benzyl-aD -glucopyranosyl)-(1 4)-2,3,6-triO -benzyl-a,b-D-glucopyranosyl trichloroacetimidate were coupled by a general condensation reaction to form the per-O-benzylated branched hexasaccharide phenyl thioglycoside. The phenylthio group of this compound was converted into a trichloroacetimidate, which was coupled with phenyl O-(2,3,6-triO -benzyl-aD -glucopyranosyl)-(1 4)-O-(2,3,6-triO -benzyl-aD -glucopyranosyl)-(1 4)-2,3,6-triO -benzyl-1-thio-b-D-glucopyranoside to afford the per-O-benzylated branched nonasaccharide phenyl thioglycoside. Replacement of the phenylthio group with a free OH-group followed by hydrogenolysis gave the desired product. The synthons reported for this synthesis constitute a versatile tool for the chemical synthesis of other complex carbohydrates.
Carbohydrate Research, Nov 1, 1995
ABSTRACT
Nature Chemistry, Jun 26, 2023
Medicinal plants are essential for improving human health, and around 75% of the population in de... more Medicinal plants are essential for improving human health, and around 75% of the population in developing countries relies mainly on herb-based medicines for health care. As the king of herb plants, ginseng has been used for nearly 5000 years in the oriental and recently in western medicines. Among the compounds studied in ginseng plants, ginsenosides have been shown to have multiple medical effects such as anti-oxidative, antiaging, anti-cancer, adaptogenic and other health-improving activities. Ginsenosides belong to a group of triterpene saponins (also called ginseng saponins) that are found almost exclusively in Panax species and accumulated especially in the plant roots. In this review, we update the conserved and diversified pathway/enzyme biosynthesizing ginsenosides which have been presented. Particularly, we highlight recent milestone works on functional characterization of key genes dedicated to the production of ginsenosides, and their application in engineering plants and yeast cells for large-scale production of ginsenosides.
Carbohydrate Research, Jul 1, 2014
The allelochemical alliarinoside present in garlic mustard (Alliaria petiolata), an invasive plan... more The allelochemical alliarinoside present in garlic mustard (Alliaria petiolata), an invasive plant species in North America, was chemically synthesized using an efficient and practical synthetic strategy based on a simple reaction sequence. Commercially available 1,2,3,4,6-penta-O-acetyl-β-D-glucopyranose was converted into prop-2-enyl 2&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;,3&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;,4&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;,6&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-tetra-O-acetyl-β-D-glucopyranoside and subjected to epoxidation. In a one-pot reaction, ring-opening of the epoxide using TMSCN under solvent free conditions followed by treatment of the formed trimethylsilyloxy nitrile with pyridine and phosphoryl chloride, afforded the acetylated β-unsaturated nitriles (Z)-4-(2&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;,3&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;,4&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;,6&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-tetra-O-β-D-glucopyranosyloxy)but-2-enenitrile and its isomer (E)-4-(2&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;,3&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;,4&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;,6&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-tetra-O-β-D-glucopyranosyloxy)but-2-enenitrile. Deacetylation of Z- and/or E-isomers afforded the target molecules alliarinoside and its isomer.
Carbohydrate Research, 2003
A short and practical methodology for conversion of unprotected D-glucose, maltose, cellobiose an... more A short and practical methodology for conversion of unprotected D-glucose, maltose, cellobiose and lactose into the corresponding phenyl 4,6-O-benzylidine-per-O-acetylated-1,2-trans-thioglycosides is described. The protocol is based on the execution of five reaction steps (bromoacetylation, thiophenolysis under phase transfer catalysis conditions, deacetylation, benzylidenation and acetylation) in one continuous procedure and provides a fast access to the title compounds as pure crystalline products without chromatographic purification.
Phytochemistry, May 1, 2012
Lotus japonicus contains the two cyanogenic glucosides, linamarin and lotaustralin, and the non c... more Lotus japonicus contains the two cyanogenic glucosides, linamarin and lotaustralin, and the non cyanogenic hydroxynitriles, rhodiocyanoside A and D, with rhodiocyanoside A as the major rhodiocyanoside. Rhodiocyanosides are structurally related to cyanogenic glucosides but are not cyanogenic. In vitro administration of intermediates of the lotaustralin pathway to microsomes prepared from selected L. japonicus accessions identified 2-methyl-2-butenenitrile as an intermediate in the rhodiocyanoside biosynthetic pathway. In vitro inhibitory studies with carbon monoxide and tetcyclacis indicate that the conversion of (Z)-2-methylbutanal oxime to 2-methyl-2-butenenitrile is catalyzed by cytochrome P450(s). Carbon monoxide inhibited cyanogenic glucosides as well as rhodiocyanosides synthesis, but inhibition of the latter pathway was much stronger. These results demonstrate that the cyanogenic glucoside and rhodiocyanosides pathways share CYP79Ds to obtain (Z)-2-methylbutanaloxime from l-isoleucine, whereas the subsequent conversions are catalyzed by different P450s. The aglycon of rhodiocyanoside A forms the cyclic product 3-methyl-2(5H)-furanone. Furanones are known to possess antimicrobial properties indicating that rhodiocyanoside A may have evolved to serve as a phytoanticipin that following β-glucosidase activation and cyclization of the aglycone formed, give rise to a potent defense compound.
Plant Journal, Dec 1, 2009
Traditional methods to localize b-glycosidase activity in tissue sections have been based on incu... more Traditional methods to localize b-glycosidase activity in tissue sections have been based on incubation with the general substrate 6-bromo-2-naphthyl-b-D-glucopyranoside. When hydrolysed in the presence of salt zinc compounds, 6-bromo-2-naphthyl-b-D-glucopyranoside affords the formation of an insoluble coloured product. This technique does not distinguish between different b-glycosidases present in the tissue. To be able to monitor the occurrence of individual b-glycosidases in different tissues and cell types, we have developed a versatile histochemical method that can be used for localization of any b-glycosidase that upon incubation with its specific substrate releases a reducing sugar. Experimentally, the method is based on hydrolysis of the specific substrate followed by oxidation of the sugar released by a tetrazolium salt (2,3,5-triphenyltetrazolium chloride) that forms a red insoluble product when reduced. The applicability of the method was demonstrated by tissue and cellular localization of two b-glucosidases, amygdalin hydrolase and prunasin hydrolase, in different tissues and cell types of almond. In those cases where the analysed tissue had a high content of reducing sugars, this resulted in strong staining of the background. This interfering staining of the background was avoided by prior incubation with sodium borohydride. The specificity of the devised method was demonstrated in a parallel localization study using a specific antibody towards prunasin hydrolase.
Scientific Reports, Nov 14, 2016
Genomic gene clusters for the biosynthesis of chemical defence compounds are increasingly identif... more Genomic gene clusters for the biosynthesis of chemical defence compounds are increasingly identified in plant genomes. We previously reported the independent evolution of biosynthetic gene clusters for cyanogenic glucoside biosynthesis in three plant lineages. Here we report that the gene cluster for the cyanogenic glucoside dhurrin in Sorghum bicolor additionally contains a gene, SbMATE2, encoding a transporter of the multidrug and toxic compound extrusion (MATE) family, which is co-expressed with the biosynthetic genes. The predicted localisation of SbMATE2 to the vacuolar membrane was demonstrated experimentally by transient expression of a SbMATE2-YFP fusion protein and confocal microscopy. Transport studies in Xenopus laevis oocytes demonstrate that SbMATE2 is able to transport dhurrin. In addition, SbMATE2 was able to transport non-endogenous cyanogenic glucosides, but not the anthocyanin cyanidin 3-O-glucoside or the glucosinolate indol-3-yl-methyl glucosinolate. The genomic co-localisation of a transporter gene with the biosynthetic genes producing the transported compound is discussed in relation to the role self-toxicity of chemical defence compounds may play in the formation of gene clusters.
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Papers by Mohammed Motawia