The Cloning, Characterisation and Antisense Cassettes Construction for Some Pigmentation Genes from Oncidium SPP.

Genes that are responsible for color formation within the anthocyanin and carotenoid biosynthesis pathway have been isolated, studied and manipulated to produce novel colors in many plant species. Therefore, in this study, the genes that are involved in flower color formation namely CHS, F3H, DFR and PSY will be isolated and studied in the orchid species, Oncidium goldiana. The construction of antisense cassettes will also be carried out for CHS, DFR and PSY. RNA was isolated from various stages of Oncidium go/diana and its purity and concentration was determined. Partial genes of CHS (605bp), F3H (503bp), DFR (415bp) and PSY (543bp) was isolated from flower petals by RT-PCR using degenerate primers. A BLAST search for homology revealed that these sequences had high homology at both the amino and nucleic acid levels with other known plantThe temporal and spatial gene expression studies using semi-quantitative RT -PCR showed that F3H and DFR were floral specific and they had the highest expression at the yellow buds stage. The level of CHS was the highest in partially opened flowers and PSY was expressed at its highest levels in green buds. CHS and PSY were not floral specific as they were also present in leaves. The antisense cassettes were constructed for CHS, DFR and PSY. These vectors were driven by a 35S promoter and Nos 3' terminator sequence. The CHS vector was 13'480bp, the DFR was 13380bp and PSY was 13430bp. These antisense cassettes were suitable for transformation by biolistic gun bombardment or via Agrobacleriunt infectio

N-acetyldopamine quinone methide/1,2-dehydro-N-acetyl dopamine tautomerase A new enzyme involved in sclerotization of insect cuticle

AbstractThe enzyme system causing the side chain desaturation of the sclerotizing precursor, N-acetyldopamine (NADA), was solubilized from the larval cuticle of Sarcophaga bullata and resolved into three components. The first enzyme, phenoloxidase, catalyzed conversion of NADA to NADA quinone and provided it for the second enzyme (NADA quinone isomerase), which makes the highly unstable NADA quinone methide. Quinone methide was hydrated rapidly and nonenzymatically to form N-acetylnorepinephrine. In addition, it also served as the substrate for the last enzyme, quinone methide tautomerase, which converted it to 1,2-dehydro-NADA. Reconstitution of NADA side chain desaturase activity was achieved by mixing the last enzyme fraction with NADA quinone isomerase, obtained from the hemolymph of the same organism, and mushroom tyrosinase. Therefore, NADA side chain desaturation observed in insects is caused by the combined action of three enzymes rather than the action of a single specific NADA desaturase, as previously thought

The crosslinking and antimicrobial properties of tunichrome

Tunichromes are small peptides containing one or more dehydrodopa derived units that have been identified in the blood cells of at least eleven species of tunicates. Incubation of tunichromes isolated from Ascidia nigra hemocytes (or model dopa-containing compounds) under oxidative conditions with either lysozyme, cytochrome c or ovalbumin resulted in a time-dependent polymerization of these test proteins to dimers, trimers, tetramers and potentially to other oligomers. These results indicate that the oxidation products of tunichromes possess inherent crosslinking properties. Hence it is possible that tunichromes participate in tunic production by forming adducts and crosslinks with structural proteins and/or carbohydrate polymers, similar to the well-understood process of insect cuticle hardening. Since such crosslinking potentials could also be beneficial for defense reactions against invading microorganisms, antibacterial activity of tunichromes was tested using both a radial diffusion assay and the Microtox® test. Tunichromes exhibited antimicrobial activity against gram-negative bacteria Escherichia coli and Photobacterium phosphorium. However, they did not show any antimicrobial activity against the gram-positive bacteria Staphylococcus aureus at the concentrations tested. We propose that the crosslinking and antimicrobial functions are both based on the reactivity of dehydrodopa units present in the tunichromes, and their subsequent ability to form highly reactive quinone methides

Bioactivity and stability studies of anthocyanin-containing extracts from Garcinia mangostana L. and Etlingera elatior Jack

Anthocyanin-containing extract (ACE) were prepared from the pericarp of Garcinia mangostana L. (mangosteen) and the inflorescence part of Etlingera elatior Jack (torch ginger) by using acidified methanol as extraction solvent. Our present study focuses on the antioxidant and cytotoxic activity and the effect of temperature, light and pH on stability of ACE derived from G. mangostana and E. elatior. The monomeric anthocyanin and total phenolic content in E. elatior was higher (43.42±0.01 mg/L dan 10.07±0.01 gGAE /100 g) compared to G. mangostana (15.03±0.01 mg/L and 3.29±0.01 gGAE/100 g). Both ACE of E. elatior and G. mangostana exhibited free radical scavenger activity, with IC50 value of14.90±1.02 mg/mL and 15.50±0.52 mg/mL, respectively, in 2,2’-diphenyl-1-picrylhydrazyl assay. ACE of E. elatior was also found to be a good reducing and possessed higher absorbance values at concentrations range from 1.25 to 20 mg/mL in FRAP assay. ACE of E. elatior exhibited mild cytotoxicity on human ovarian SKOV-3 cell line with IC50 values of 54.32 ± 4.60 μg/mL. The effects of light and temperature on ACE stability were performed in different environmental conditions, which promote the destabilization of anthocyanin molecules. ACE stability of G. mangostana were less resist to the effect of light but very susceptible to the prolonged effect of heat after 2 h exposure compared to the ACE in E. elatior. The different in pH highly influence the stability of both ACE which can be observed from the decrease in absorbance readings

Artocarpus heterophyllus Lam. stem bark inhibits melanogenesis through regulation of ROS, cAMP, and MAPK Pathways

Natural-based skin-lightening cosmeceutical products are attracting high popularity nowadays due to their relatively high bioavailability upon application. Artocarpus species have been highlighted with such potential, and our previous studies have reported that Artocarpus heterophyllus Lam. stem bark extract exhibited a potent anti-melanogenic activity by reducing melanin content and inhibiting cellular tyrosinase activity in B16F10 melanoma cells. Hence, this study aimed to identify the bioactive fraction from A. heterophyllus Lam. stem bark and determine its anti-melanogenic mechanisms in B16F10 melanoma cells. Our results showed that a fraction (H-3) demonstrated the most pronounced anti-melanogenic effect at 12.00 µg/mL by reducing melanin content to 22.86 ± 2.90% and inhibiting cellular tyrosinase activity at treatment concentration 33-fold lower than kojic acid, without being cytotoxic against B16F10 melanoma cells. Moreover, treatment with H-3 for 24 and 48 h substantially scavenged intracellular reactive oxygen species (ROS) of hydrogen peroxide-challenged B16F10 melanoma cells by 1.8 and 4.4%, respectively. Based on the microarray profiling and qPCR analysis, H-3 downregulated Creb3l1, Creb3l2, Creb3l3, Mitf, Tyr, Tyrp1, and Dct genes in B16F10 melanoma cells, whereas the expression of Map3k20, Mapk14 (p38), and Foxo3 genes was markedly increased. Altogether, these results demonstrated that H-3 exhibited its anti-melanogenic activity in B16F10 melanoma cells through scavenging ROS and concurrent inhibition of the cAMP and activation of the p38/MAPK signaling pathways. These findings indicate that H-3 has the potential to be used as a skin lightening cosmeceutical agent in the treatment of skin hyperpigmentation

Bioactive Dehydrotyrosyl and Dehydrodopyl Compounds of Marine Origin

The amino acid, tyrosine, and its hydroxylated product, 3,4-dihydroxyphenylalanine (dopa), plays an important role in the biogenesis of a number of potentially important bioactive molecules in marine organisms. Interestingly, several of these tyrosyl and dopa-containing compounds possess dehydro groups in their side chains. Examples span the range from simple dehydrotyrosine and dehydrodopamines to complex metabolic products, including peptides and polycyclic alkaloids. Based on structural information, these compounds can be subdivided into five categories: (a) Simple dehydrotyrosine and dehydrotyramine containing molecules; (b) simple dehydrodopa derivatives; (c) peptidyl dehydrotyrosine and dehydrodopa derivatives; (d) multiple dehydrodopa containing compounds; and (e) polycyclic condensed dehydrodopa derivatives. These molecules possess a wide range of biological activities that include (but are not limited to) antitumor activity, antibiotic activity, cytotoxicity, antioxidant activity, multidrug resistance reversal, cell division inhibition, immunomodulatory activity, HIV-integrase inhibition, anti-viral, and anti-feeding (or feeding deterrent) activity. This review summarizes the structure, distribution, possible biosynthetic origin, and biological activity, of the five categories of dehydrotyrosine and dehydrodopa containing compounds

Growth Inhibition of Human Gynecologic and Colon Cancer Cells by Phyllanthus watsonii through Apoptosis Induction

Phyllanthus watsonii Airy Shaw is an endemic plant found in Peninsular Malaysia. Although there are numerous reports on the anti cancer properties of other Phyllanthus species, published information on the cytotoxicity of P. watsonii are very limited. The present study was carried out with bioassay-guided fractionation approach to evaluate the cytotoxicity and apoptosis induction capability of the P. watsonii extracts and fractions on human gynecologic (SKOV-3 and Ca Ski) and colon (HT-29) cancer cells. P. watsonii extracts exhibited strong cytotoxicity on all the cancer cells studied with IC50 values of ≤ 20.0 µg/mL. Hexane extract of P. watsonii was further subjected to bioassay-guided fractionation and yielded 10 fractions (PW-1→PW-10). PW-4→PW-8 portrayed stronger cytotoxic activity and was further subjected to bioassay-guided fractionation and resulted with 8 sub-fractions (PPWH-1→PPWH-8). PPWH-7 possessed greatest cytotoxicity (IC50 values ranged from 0.66 – 0.83 µg/mL) and was selective on the cancer cells studied. LC-MS/MS analysis of PPWH-7 revealed the presence of ellagic acid, geranic acid, glochidone, betulin, phyllanthin and sterol glucoside. Marked morphological changes, ladder-like appearance of DNA and increment in caspase-3 activity indicating apoptosis were clearly observed in both human gynecologic and colon cancer cells treated with P. watsonii especially with PPWH-7. The study also indicated that P. watsonii extracts arrested cell cycle at different growth phases in SKOV-3, Ca Ski and HT-29 cells. Cytotoxic and apoptotic potential of the endemic P. watsonii was investigated for the first time by bioassay-guided approach. These results demonstrated that P. watsonii selectively inhibits the growth of SKOV-3, Ca Ski and HT-29 cells through apoptosis induction and cell cycle modulation. Hence, P. watsonii has the potential to be further exploited for the discovery and development of new anti cancer drugs

Critical Analysis of the Melanogenic Pathway in Insects and Higher Animals

Animals synthesize melanin pigments for the coloration of their skin and use it for their protection from harmful solar radiation. Insects use melanins even more ingeniously than mammals and employ them for exoskeletal pigmentation, cuticular hardening, wound healing and innate immune responses. In this review, we discuss the biochemistry of melanogenesis process occurring in higher animals and insects. A special attention is given to number of aspects that are not previously brought to light: (1) the molecular mechanism of dopachrome conversion that leads to the production of two different dihydroxyindoles; (2) the role of catecholamine derivatives other than dopa in melanin production in animals; (3) the critical parts played by various biosynthetic enzymes associated with insect melanogenesis; and (4) the presence of a number of important gaps in both melanogenic and sclerotinogenic pathways. Additionally, importance of the melanogenic process in insect physiology especially in the sclerotization of their exoskeleton, wound healing reactions and innate immune responses is highlighted. The comparative biochemistry of melanization with sclerotization is also discussed

Reactivities of Quinone Methides versus o-Quinones in Catecholamine Metabolism and Eumelanin Biosynthesis

Melanin is an important biopolymeric pigment produced in a vast majority of organisms. Tyrosine and its hydroxylated product, dopa, form the starting material for melanin biosynthesis. Earlier studies by Raper and Mason resulted in the identification of dopachrome and dihydroxyindoles as important intermediates and paved way for the establishment of well-known Raper–Mason pathway for the biogenesis of brown to black eumelanins. Tyrosinase catalyzes the oxidation of tyrosine as well as dopa to dopaquinone. Dopaquinone thus formed, undergoes intramolecular cyclization to form leucochrome, which is further oxidized to dopachrome. Dopachrome is either converted into 5,6-dihydroxyindole by decarboxylative aromatization or isomerized into 5,6-dihydroxyindole-2-carboxylic acid. Oxidative polymerization of these two dihydroxyindoles eventually produces eumelanin pigments via melanochrome. While the role of quinones in the biosynthetic pathway is very well acknowledged, that of isomeric quinone methides, however, remained marginalized. This review article summarizes the key role of quinone methides during the oxidative transformation of a vast array of catecholamine derivatives and brings out the importance of these transient reactive species during the melanogenic process. In addition, possible reactions of quinone methides at various stages of melanogenesis are discussed

A systematic study of Fagraea Sensu Lato and a revision of the Peninsular Malaysian Taxa / Sugumaran Manickam

systematic study of the Peninsular Malaysian species of Fagraea sensu lato was undertaken. This was done in the light of recent revisionary work done for Borneo that documented 20 new species and demonstrated that previous species concepts for Fagraea were too broad. Parallel to this the distinction of the subgeneric groups recognised as sections, i.e., Cyrtophyllum, Fagraea and Racemosae, were investigated with molecular phylogenetic methods. Representative taxa from the Malay Peninsula and Borneo augmented by sequences from other taxa in the same subtribe and tribe were used in the molecular analysis. Gene sequences from ITS, trnL–F (trnL intron + trnL–F spacer) and ndhF were analysed with two methods viz., maximum parsimony and Bayesian analyses. The results indicate that Fagraea s.l. includes four well-supported monophyletic groups, with the several gene sequences analysed with two phylogenetic methods. Two of the clades, viz., Fagraea and Racemosa, could be equated to sections Fagraea and Racemosae, respectively. The remaining two clades, viz., Elliptica and Gigantea appear to be parts of the section Cyrtophyllum. The Racemosa clade had the most morphological synapomorphies, with a distinct plant architecture where trunk growth is continuous and branches are plagiotropic (with distichous leaf arrangement); pendulous inflorescences; and a firm fruit wall with an epidermis that does not detach and wrinkle upon drying. The Fagraea clade (excluding Fagraea crenulata) has fruits that produce copious creamy pale yellowish latex in the fruit epidermis and fruit wall and have ellipsoid-rounded seeds. In comparison, all the other species of Fagraea (including F. crenulata) either have no latex or produce small amounts of translucent gummy latex and have polygonal seeds. F. crenulata is aberrant in the Fagraea clade in having unique characters such as a distinct architectural model, thorny bark and crenulate leaf margins. It is however, related to the Fagraea clade in having petiolar sheaths that do not or only slightly fuse at the edges and a peltate stigma structure. Phylogenetic analyses with the ITS region did not include F. crenulata in the Fagraea clade. However, F. crenulata is resolved basal to the Fagraea clade with chloroplast gene analyses. The clear split of section Cyrtophyllum into the Gigantea and Elliptica clades was somewhat surprising as these groups have a number of similar morphological features, such as small flowers and muchprotruding stamens and styles. In comparison, the other groups are generally distinguishable with bigger flowers and less exserted stamens and styles. The only morphological difference between these two groups is the position of the inflorescence, terminal in Elliptica and axillary in Gigantea. Recognition of Fagraea s.l. as four distinct genera is indicated, as the complex is considered morphologically too divergent to be regarded as a single genus. These correspond to the four clades recognised in the molecular analyses, viz., Elliptica, Fagraea, Gigantea and Racemosa, and could adopt Picrophloeus Bl., Fagraea Thunb. (sensu stricto), Cyrtophyllum Reinw. ex Bl. and Utania G.Don, respectively, as good genus names. The position of F. crenulata is doubtful and it is provisionally maintained in Fagraea s.s. pending future molecular investigations with a larger taxon sampling over wider geographical context, and the use of further gene regions