Analysis of capsaicinoid biosynthesis pathway genes expression in callus cultures of Capsicum chinense Jacq. cv. ‘Umorok’

The placental tissue of the highly pungent chilli cultivar, Capsicum chinense Jacq. cv. ‘Umorok’, is used as explants for callus induction. Callus cultures were subcultured after every 32 days and growth curves for a period of six consecutive growth cycles were studied till a stable capsaicinoids producing callus cultures were obtained. The capsaicinoids content in placental tissue explants decreased gradually during the first 2 months of culture as the explants dedifferentiated to form friable callus while the biomass and capsaicinoid content did not show much change in the subsequent growth cycles. The maximum callus biomass of 7.8 g freshweight (FW) or 0.56 g dry weight (DW) per culture were obtained on the 24th day of every growth cycle and the maximum average capsaicinoids content (1.6 mg g^?1 FW capsaicin and 0.78 mg g^?1 FW dihydrocapsaicin) were obtained on the 20th day of every growth cycle. To investigate the underlying dynamics for capsaicinoid biosynthesis during callus formation, comparative gene expression analysis of the genes involved in capsaicinoid biosynthesis pathway were also studied by qRT-PCR analysis. When compared with placental tissue, all the studied genes showed reduced expression during callus formation, especially putative aminotransferase (pAMT) and pungent gene 1 (Pun1), which were extensively down regulated from the 3rd month onwards in the callus cultures. Therefore, the present study revealed that the down-regulated expression of mainly two putative genes in capsaicinoid biosynthetic pathway (pAMT and Pun1) resulted in lower accumulation of capsaicinoids in callus cultures compared to placental tissues of fruits.

     

Haloperidol induces calcium ion influx via L-type calcium channels in hippocampal HN33 cells and renders the neurons more susceptible to oxidative stress

Haloperidol is a classical neuroleptic drug that is still in clinical use and can lead to abnormal motor activity following repeated administration. However, there is little knowledge of how it triggers neuronal impairment. In this study, we report that it induced calcium ion influx via L-type calcium channels and that the elevation of calcium ions induced by haloperidol appeared to render hippocampal cells more susceptible to oxidative stress. Indeed, the level of cytotoxic reactive oxygen species (ROS) and the expression of pro-apoptotic Bax increased in response to oxidative stress in haloperidol-treated cells, and these effects were inhibited by verapamil, a specific L-type calcium channel blocker, but not by the T-type calcium channel blocker, mibefradil. These findings indicate that haloperidol induces calcium ion influx via L-type calcium channels and that this calcium influx influences neuronal fate.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Capsicum chinense</Emphasis> Jacq.

An efficient micropropagation protocol was established for Capsicum chinense Jacq. cv. Umorok, a pungent chilli cultivar. Shoot-tip explants were cultured on Murashige and Skoog (MS) medium containing cytokinins (22.2–88.8 μM 6-benzylaminopurine, BAP, 23.2–93.0 μM kinetin, Kin, or 22.8–91.2 μM zeatin, Z) alone or in combination with 5.7 μM indole-3-acetic acid (IAA). Maximum number of shoots were induced on medium containing 91.2 μM Z or 31.1 μM BAP with 4.7 μM Kin. The separated shoots rooted and elongated on medium containing 2.5 or 4.9 μM indole-3-butyric acid (IBA). Axillary shoots were induced from in vitro raised plantlets by decapitating them. The axillary shoot-tip explants were used for further multiple shoot buds induction. A maximum of about 150 plantlets were obtained from a single seedling. Hardened and acclimatized plantlets were successfully established in the soil.     

Identification of a new functional target of haloperidol metabolite: implications for a receptor-independent role of 3-(4-fluorobenzoyl) propionic acid

Haloperidol, a dopamine D2 receptor blocker, is a classical neuroleptic drug that elicits extrapyramidal symptoms. Its metabolites include 3-(4-fluorobenzoyl) propionic acid (FBPA) and 4-(4-chlorophenyl)-4-piperidinol (CPHP). Until now, the biological significance of these metabolites has remained largely unknown. Here, we report that the administration of FBPA to mice effected a suppression of locomotor activity and induced catalepsy in a manner similar to that observed with haloperidol, whereas CPHP had no significant effects. Neither of these two metabolites, however, exhibited any ability to bind to the dopamine D2 receptor. FBPA blocked dopamine-induced extracellular signal-regulated kinase 1/2 phosphorylation, and it specifically affected mitogen-activated protein kinase kinase (MEK)1/2 activity in hippocampal HN33 cells. Moreover, FBPA was capable of direct interaction with MEK1/2, and inhibited its activity in vitro. We demonstrated the generation of haloperidol metabolites within haloperidol-treated cells by mass spectrometric analyses. Collectively, our results confirm the biological activity of FBPA, and provide initial clues as to the receptor-independent role of haloperidol.     

<Emphasis Type="Italic">In vitro</Emphasis> plant regeneration in six cultivars of <Emphasis Type="Italic">Capsicum</Emphasis> spp. using different explants

In vitro regeneration from leaf, cotyledon and hypocotyl explants of six cultivars belonging to three species of Capsicum was achieved by direct organogenesis. The cultivar Umorok showed the best response while Meiteimorok, Haomorok, Mashingkha and Uchithi showed intermediate response and the cultivar Chiengpi was the least responsive. Leaf and cotyledon explants regenerated more shoots than hypocotyl explants and the maximum number of shoots were produced on Murashige and Skoog (1962) medium containing 8.8 μM 6-benzylaminopurine (BAP) with 11.4 μM indole-3-acetic acid (IAA). Elongation of shoot buds derived from different explants was achieved on medium containing 2.8 μM IAA and the elongated shoots were rooted on medium containing 2.8 or 5.7 μM IAA and 2.4 or 4.9 μM indole-3-butyric acid (IBA). Four-week old rooted plantlets were hardened and transplanted to the soil. The plantlets showed 90 % survival during transplantation.     

Occurrence of Cotton leaf curl Multan virus and associated betasatellites with leaf curl disease of Bhut-Jolokia chillies (Capsicum chinense Jacq.) in India

Molecular Biology Reports - Geminiviridae comprises the largest family of plant viruses which causes severe crop losses in India. The highest pungency chilli Bhut-Jolokia or ghost pepper (Capsicum...