Identification of candidate genes involved in steroidal alkaloids biosynthesis in organ-specific transcriptomes of Veratrum nigrum L.

Veratrum nigrum is protected plant of Melanthiaceae family, able to synthetize unique steroidal alkaloids important for pharmacy. Transcriptomes from leaves, stems and rhizomes of in vitro maintained V. nigrum plants were sequenced and annotated for genes and markers discovery. Sequencing of samples derived from the different organs resulted in a total of 108,511 contigs with a mean length of 596 bp. Transcripts derived from leaf and stalk were annotated at 28%, and 38% in Nr nucleotide database, respectively. The sequencing revealed 949 unigenes related with lipid metabolism, including 73 transcripts involved in steroids and genus-specific steroid alkaloids biosynthesis. Additionally, 3203 candidate SSRs markers we identified in unigenes with average density of one SSR locus every 6.2 kb sequence. Unraveling of biochemical machinery of the pathway responsible for steroidal alkaloids will open possibility to design and optimize biotechnological process. The transcriptomic data provide valuable resources for biochemical, molecular genetics, comparative transcriptomics, functional genomics, ecological and evolutionary studies of V. nigrum.

Dietary antioxidants as a source of hydrogen peroxide.

Studies of 54 antioxidants revealed that 27 of them, mainly polyphenols, generated hydrogen peroxide (H2O2) when added to Dulbecco's modified Eagle's medium (DMEM), other media used for culture of mammalian and yeast cells and phosphate-buffered saline. The most active antioxidants were: propyl gallate (PG), (-)-epigallocatechin gallate (EGCG) and quercetin (Q). Chelex treatment and iron chelators decreased H2O2 generation suggesting that transition metal ions catalyze antioxidant autoxidation and H2O2 production. Green tea also generated H2O2; tea prepared on tap water generated significantly more H2O2 than tea prepared on deionized water. Ascorbic acid decreased H2O2 production although it generated H2O2 itself, in the absence of other additives. Lemon added to the tea significantly reduced generation of H2O2. Hydrogen peroxide generated in the medium contributed to the cytotoxicity of PG, EGCG and Q to human prostate carcinoma DU-145 cells, since catalase increased the survival of the cells subjected to these compounds in vitro.

Antioxidant properties of ferrous flavanol mixtures.

Interaction of metal, especially iron ions with flavanols is considered as an important feature of these compounds and is believed to contribute to their both antioxidant and prooxidant properties. The aim of this study was to examine how Fe2+ binding to form a 4:1 (flavanol:Fe2+) mixtures affects the antioxidant properties of flavanols. ABTS∗ scavenging, protection against fluorescence bleaching induced by AAPH and hypochlorite, protection against lipid peroxidation and protection against hypochlorite-induced hemolysis demonstrated that flavonol-Fe2+ mixtures retain antioxidant properties, although, in most cases, they are lower with respect to the flavanols alone. No superoxide dismutase-like or catalase-like activity of the mixtures was revealed.

Origin and pathophysiology of protein carbonylation, nitration and chlorination in age-related brain diseases and aging.

Non-enzymatic protein modifications occur inevitably in all living systems. Products of such modifications accumulate during aging of cells and organisms and may contribute to their age-related functional deterioration. This review presents the formation of irreversible protein modifications such as carbonylation, nitration and chlorination, modifications by 4-hydroxynonenal, removal of modified proteins and accumulation of these protein modifications during aging of humans and model organisms, and their enhanced accumulation in age-related brain diseases.

Antioxidant properties of catechins: Comparison with other antioxidants.

Antioxidant properties of five catechins and five other flavonoids were compared with several other natural and synthetic compounds and related to glutathione and ascorbate as key endogenous antioxidants in several in vitro tests and assays involving erythrocytes. Catechins showed the highest ABTS-scavenging capacity, the highest stoichiometry of Fe3+ reduction in the FRAP assay and belonged to the most efficient compounds in protection against SIN-1 induced oxidation of dihydrorhodamine 123, AAPH-induced fluorescein bleaching and hypochlorite-induced fluorescein bleaching. Glutathione and ascorbate were less effective. (+)-catechin and (-)-epicatechin were the most effective compounds in protection against AAPH-induced erythrocyte hemolysis while (-)-epicatechin gallate, (-)-epigallocatechin gallate and (-)-epigallocatechin protected at lowest concentrations against hypochlorite-induced hemolysis. Catechins [(-)-epigallocatechin gallate and (-)-epicatechin gallate)] were most efficient in the inhibition of AAPH-induced oxidation of 2'7'-dichlorodihydroflurescein contained inside erythrocytes. Excellent antioxidant properties of catechins and other flavonoids make them ideal candidates for nanoformulations to be used in antioxidant therapy.

Oxidative Modification of Blood Serum Proteins in Multiple Sclerosis after Interferon Beta and Melatonin Treatment.

Multiple sclerosis (MS) is a disease involving oxidative stress (OS). This study was aimed at examination of the effect of melatonin supplementation on OS parameters, especially oxidative protein modifications of blood serum proteins, in MS patients. The study included 11 control subjects, 14 de novo diagnosed MS patients with the relapsing-remitting form of MS (RRMS), 36 patients with RRMS receiving interferon beta-1b (250 µg every other day), and 25 RRMS patients receiving interferon beta-1b plus melatonin (5 mg daily). The levels of N'-formylkynurenine, kynurenine, dityrosine, carbonyl groups, advanced glycation products (AGEs), advanced oxidation protein products (AOPP), and malondialdehyde were elevated in nontreated RRSM patients. N'-Formylkynurenine, kynurenine, AGEs, and carbonyl contents were decreased only in the group treated with interferon beta plus melatonin, while dityrosine and AOPP contents were decreased both in the group of patients treated with interferon beta and in the group treated with interferon beta-1b plus melatonin. These results demonstrate that melatonin ameliorates OS in MS patients supporting the view that combined administration of interferon beta-1b and melatonin can be more effective in reducing OS in MS patients than interferon beta-1b alone.

Changes in fenugreek transcriptome induced by methyl jasmonate and steroid precursors revealed by RNA-Seq.

Changes in fenugreek transcriptome related to enhanced production of steroids were induced by methyl jasmonate, cholesterol and squalene, and recorded using RNA-seq. A total of 112,850 unigenes were obtained after de novo assembling of next generation sequencing data, and used for functional annotations. In steroidal saponins pathway, transcripts involved in mevalonate, terpenoid backbone and plant sterol synthesis were annotated. Overexpression of several transcripts from phytosterol biosynthesis pathway was confirmed by quantitative RT-PCR. In diosgenin biosynthesis pathway, fatty acid ω-hydroxylase (CYP86A2) and steroid 22-alpha-hydroxylase (CYP90B1) genes were annotated in all induced transcriptomes. Moreover, direct sequencing confirmed increased levels of CYP90B1, unspecific monooxygenase and 26-hydroxylase genes in plants with elevated level of diosgenin. New unigenes corresponding to enzymes involved in biosynthesis of diosgenin from cycloartenol via cholesterol were obtained and the role of CYP72A family in steroidal saponin biosynthesis was proposed. Additional support for biosynthetic pathway from cycloartenol to diosgenin was provided.

Next-generation sequencing of representational difference analysis products for identification of genes involved in diosgenin biosynthesis in fenugreek (Trigonella foenum-graecum).

MAIN CONCLUSION: Representational difference analysis of cDNA was performed and differential products were sequenced and annotated. Candidate genes involved in biosynthesis of diosgenin in fenugreek were identified. Detailed mechanism of diosgenin synthesis was proposed. Fenugreek (Trigonella foenum-graecum L.) is a valuable medicinal and crop plant. It belongs to Fabaceae family and has a unique potential to synthesize valuable steroidal saponins, e.g., diosgenin. Elicitation (methyl jasmonate) and precursor feeding (cholesterol and squalene) were used to enhance the content of sterols and steroidal sapogenins in in vitro grown plants for representational difference analysis of cDNA (cDNA-RDA). To identify candidate genes involved in diosgenin biosynthesis, differential, factor-specific libraries were subject to the next-generation sequencing. Approximately 9.9 million reads were obtained, trimmed, and assembled into 31,491 unigenes with an average length of 291 bp. Then, functional annotation and gene ontogeny enrichment analysis was performed by aligning all-unigenes with public databases. Within the transcripts related to sterol and steroidal saponin biosynthesis, we discovered novel candidate genes of diosgenin biosynthesis and validated their expression using quantitative RT-PCR analysis. Based on these findings, we supported the idea that diosgenin is biosynthesized from cycloartenol via cholesterol. This is the first report on the next-generation sequencing of cDNA-RDA products. Analysis of the transcriptomes enriched in low copy sequences contributed substantially to our understanding of the biochemical pathways of steroid synthesis in fenugreek.