In Vitro and In Vivo Characterization of the Alkaloid Nuciferine

Rationale

The sacred lotus (Nelumbo nucifera) contains many phytochemicals and has a history of human use. To determine which compounds may be responsible for reported psychotropic effects, we used in silico predictions of the identified phytochemicals. Nuciferine, an alkaloid component of Nelumbo nucifera and Nymphaea caerulea, had a predicted molecular profile similar to antipsychotic compounds. Our study characterizes nuciferine using in vitro and in vivo pharmacological assays.

Methods

Nuciferine was first characterized in silico using the similarity ensemble approach, and was followed by further characterization and validation using the Psychoactive Drug Screening Program of the National Institute of Mental Health. Nuciferine was then tested in vivo in the head-twitch response, pre-pulse inhibition, hyperlocomotor activity, and drug discrimination paradigms.

Results

Nuciferine shares a receptor profile similar to aripiprazole-like antipsychotic drugs. Nuciferine was an antagonist at 5-HT_2A, 5-HT_2C, and 5-HT_2B, an inverse agonist at 5-HT₇, a partial agonist at D_2, D₅ and 5-HT₆, an agonist at 5-HT_1A and D₄ receptors, and inhibited the dopamine transporter. In rodent models relevant to antipsychotic drug action, nuciferine blocked head-twitch responses and discriminative stimulus effects of a 5-HT_2A agonist, substituted for clozapine discriminative stimulus, enhanced amphetamine induced locomotor activity, inhibited phencyclidine (PCP)-induced locomotor activity, and rescued PCP-induced disruption of prepulse inhibition without induction of catalepsy.

Conclusions

The molecular profile of nuciferine was similar but not identical to that shared with several approved antipsychotic drugs suggesting that nuciferine has atypical antipsychotic-like actions.     

Studies on the microflora associated with xenic cultures of Entamoeba gingivalis

The microflora associated with xenic stock cultures (ATCC 30927) of Entamoeba gingivalis, the major protozoan of the human oral cavity, were isolated and identified as Citrobacter diversus, Yersinia enterocolitica, Acinetobacter anitratus and Pseudomonas maltophilia. In studies to determine whether the bacterial isolates were able to utilize rice starch as a sole carbon source, Y. enterocolitica exhibited excellent growth in rice starch minimal medium and TYSGM-9 medium (with rice starch), but growth was weak in TYSGM-9 medium (without rice starch). C. diversus, A. anitratus and P. maltophilia exhibited poor growth in rice starch minimal medium, but they produced excellent growth in TYSGM-9 medium with or without rice starch. In order to determine the effect of the rice starch hydrolysis on Entamoeba growth, the filtrate from each isolate grown in rice starch minimal medium was added to an E. gingivalis culture grown in TYSGM-9 medium. The filtrate from a Y. enterocolitica culture grown in rice starch minimal medium enhanced E. gingivalis growth, but the filtrates from cultures of C. diversus, A. anitratus and P. maltophilia suppressed E. gingivalis growth. This supported the concept that Y. enterocolitica is capable of metabolizing rice starch into intermediate products, which in turn can be utilized by the amoeba.     

Atlantic salmon (Salmo salar) serum albumin: cDNA sequence, evolution, and tissue expression

Atlantic salmon serum albumin is one of the most abundant proteins in salmon liver, representing 1.6% of all clones in a cDNA library made from salmon liver RNA. The DNA from a number of clones was sequenced to reveal an open reading frame of 1,827 bases encoding a 608-amino-acid protein. The sequenced 5' untranslated region is 69 bases long and the 3' untranslated region contains two putative polyadenylation signals and poly(A) tail. Sequence analysis of different clones indicates the presence of a second cDNA for salmon serum albumin. Multiple alignments of salmon serum albumin deduced amino acid sequence with Xenopus laevis, rat, bovine, and human serum albumins shows significant conservation of cysteine residues. The triple domain structure of serum albumin proteins is maintained. Unlike mammalian systems where serum albumin expression appears to be specific to liver only, salmon serum albumin is expressed in muscle also.     

Growth studies on xenic cultures of Entamoeba gingivalis using established media

Wantland's egg medium, modified Shaffer-Frye (MSF) medium and Tryptose-Trypticase-Yeast Extract-Serum-Blood (TTY-SB) medium were compared with variations of the latter two media for their ability to support xenic growth of Entamoeba gingivalis. Wantland's egg medium was unsuitable for growth of E. gingivalis. Accompanying bacteria became resistant to penicillin and streptomycin, overwhelming the amoeba culture. MSF medium was also unsuitable for the cultivation of E. gingivalis. Bacterial growth was heavy and protozoan growth sparse. MSF medium without mercaptosuccinic acid, but with rice starch, dextran or levan substituted for glucose and with Yersinia enterocolitica added, supported limited growth of the amoeba. Unmodified TTY-SB medium did not sustain growth of E. gingivalis. However, when rice starch suspension was substituted for glucose, -cysteine HCl was deleted, and a Crithidia sp. was added to the E. gingivalis culture grown xenically, enhanced growth of the oral amoeba resulted in this modified TTY-SB medium. E. gingivalis is very sensitive to changes in incubation temperature. Optimum growth was found to be in the narrow range from 34.5 to 35°C for all media tested.     

An antibiotic-free medium for the xenic cultivation of Entamoeba gingivalis.

Diamond's TYI-S-33 (Trypticase-Yeast Extract-Iron-Serum) medium was used as the basis for a new antibiotic-free medium for xenic growth of Entamoeba gingivalis. Nutritional requirements of the oral protozoan were determined in an effort to optimize growth. TYI-S-33 medium did not support E. gingivalis growth prior to modification. The changes included: (a) deletion of L-cysteine.HCl and thioctic acid, (b) substitution of glucose for dextran I (mol. wt 185,000) or rice starch, (c) reduction of concentrations of tryptone (2.5 g l-1), yeast extract (1.25 g l-1) and dextran I (1 g l-1), (d) increased concentration of ferric ammonium citrate (0.2 g l-1), and (e) addition of gastric mucin (2.4 g l-1). Dextran I was chosen as the major carbon source; its use in the medium limited growth of accompanying bacteria. This new antibiotic-free medium significantly increased E. gingivalis growth (16-20 E. gingivalis trophozoites observed per field) as compared to growth in Diamond's TYSGM-9 (Trypticase-Yeast Extract-Serum-Gastric Mucin) medium (six to 10 E. gingivalis trophozoites observed per field).     

Microvascular architecture of the pregnant rabbit oviduct

Methyl-methacrylate vascular corrosion casts of the oviducts were prepared in 7 rabbits which were 2-3 weeks pregnant. Scanning electron microscopy of the acrylic casts revealed little change in tubal microvascular connections when compared with control oviducts. Venous distension in the isthmic subserosal venous plexus, ampullary subserosal vasculature and in the fimbrial core was substantially greater than that observed in controls. These changes are interpreted as indicating a sensitivity of tubal microvasculature to the increased levels of circulating placental hormones in pregnancy. The implications of this interpretation in the role of tubal microvasculature at the time of ovulation are discussed.