Fungal hallucinogens psilocin, ibotenic acid, and muscimol: analytical methods and biologic activities.

Psychoactive drugs of fungal origin, psilocin, ibotenic acid, and muscimol among them have been proposed for recreational use and popularized since the 1960s, XX century. Despite their well-documented neurotoxicity, they reached reputation of being safe and nonaddictive. Scientific efforts to find any medical application for these hallucinogens in psychiatry, psychotherapy, and even for religious rituals support are highly controversial. Even if they show any healing potential, their usage in psychotherapy is in some cases inadequate and may additionally harm seriously suffering patients. Hallucinogens are thought to reduce cognitive functions. However, in case of indolealkylamines, such as psilocin, some recent findings suggest their ability to improve perception and mental skills, what would motivate the consumption of "magic mushrooms." The present article offers an opportunity to find out what are the main symptoms of intoxication with mushrooms containing psilocybin/psilocin, muscimol, and ibotenic acid. The progress in analytical methods for detection of them in fungal material, food, and body fluids is reviewed. Findings on the mechanisms of their biologic activity are summarized. Additionally, therapeutic potential of these fungal psychoactive compounds and health risk associated with their abuse are discussed.

Liposomal formulation of 5-fluorocytosine in suicide gene therapy with cytosine deaminase--for colorectal cancer.

It is generally accepted that successful gene therapy depends on two major factors: tumor-specific expression of a therapeutic gene and the efficient transfer of a therapeutic gene to tumor cells. For gene-directed enzyme prodrug therapy (GDEPT) involving Escherichia coli cytosine deaminase (CD) and 5-fluorocytosine (5-FC), several tumor-specific promoters and virus-based vectors were used. No attention whatsoever was paid to the way of 5-FC delivery to solid tumors, despite the fact that the delivery of drugs to such tumors is generally low because of their insufficient transfer from the blood. To compare the effectiveness of GDEPT with free and liposomal 5-FC, the prodrug was encapsulated in liposomes composed of dipalmitoylphosphatidylcholine (DPPC) and cholesterol (1:1). When the liposomal form of 5-FC was administered i.v., mice treated with a dose of 5mg of liposomal 5-FC/kg body weight for 10 days, showed complete regression of transplanted tumors and complete cure was observed, whereas in animals treated with the same amounts of the free prodrug, 50% tumor regression and only insignificantly prolonged median survival were found. In summary, these results showed a remarkable enhancement of the antitumor effects of the liposomal form of 5-FC in comparison with the free prodrug. Therapy with liposomal 5-FC thus represents a new approach to achieving a high local concentration of the prodrug for suicide gene therapy using E. coli CD.

Lipid-binding role of betaII-spectrin ankyrin-binding domain.

It is known that erythroid and non-erythroid spectrins binding of vesicles and monolayers containing PE proved sensitive to inhibition by red blood cell ankyrin. We now show that the bacterially-expressed recombinant peptides representing betaII(brain)-spectrin's ankyrin-binding domain and its truncated mutants showed lipid-binding activity, although only those containing a full-length amino terminal fragment showed high to moderate affinity towards phospholipid mono- and bilayers and a substantial sensitivity of this binding to inhibition by ankyrin. These results are in accordance with our published data on betaI-spectrin's ankyrin-binding domain [Hryniewicz-Jankowska A, et al. Mapping of ankyrin-sensitive, PE/PC mono- and bilayer binding site in erythroid beta-spectrin. Biochem J 2004;382:677-85]. Moreover, we tested also the effect of transient transfection of living cells of several cell-lines with vectors coding for GFP-conjugates including betaII and also betaI full-length ankyrin-binding domain and their truncated fragments on the membrane skeleton organization. The transfection with constructs encoding full-length ankyrin-binding domain of betaII and betaI spectrin resulted in increased aggregation of membrane skeleton and its punctate appearance in contrast to near normal appearance of membrane skeleton of cells transiently transfected with GFP control or construct encoding ankyrin-binding domain truncated at their N-terminal region. Our results therefore indicate the importance of N-terminal region for lipid-binding activity of the beta-spectrin ankyrin-binding domain and its substantial role in maintaining the spectrin-based skeleton distribution.

Higly fusogenic cationic liposomes transiently permeabilize the plasma membrane of HeLa cells.

Cationic liposomes can efficiently carry nucleic acids into mammalian cells. This property is tightly connected with their ability to fuse with negatively charged natural membranes (i.e. the plasma membrane and endosomal membrane). We used FRET to monitor and compare the efficiency of lipid mixing of two liposomal preparations--one of short-chained diC14-amidine and one of long-chained unsaturated DOTAP--with the plasma membrane of HeLa cells. The diC14-amidine liposomes displayed a much higher susceptibility to lipid mixing with the target membranes. They disrupted the membrane integrity of the HeLa cells, as detected using the propidium iodide permeabilization test. Morphological changes were transient and essentially did not affect the viability of the HeLa cells. The diC14-amidine liposomes were much more effective at either inducing lipid mixing or facilitating transfection.

PS exposure increases the susceptibility of cells to fusion with DOTAP liposomes.

Cationic liposomes are used as efficient carriers for gene delivery into mammalian cells due to their ability to bind nucleic acids, adsorb onto the cell surface and fuse with negatively charged membranes. This last property enables the release and escape of their cargo from endosomal compartments. The efficiency of this fusion mainly depends on the surface charge of the target membranes. Here, we report that cells of two different lines, epithelial adenocarcinoma HeLa and lymphocytic leukemia Jurkat T, which externalize PS, are more susceptible to fusion with DOTAP liposomes than control cells. We compared the ability to undergo fusion of untreated and apoptotic cells. Apoptosis was induced by various pro-apoptotic agents and treatments, namely: incubation in the presence of MnCl(2), cytostatic drugs fludarabine and mitoxantrone, staurosporine and serum depletion in the case of HeLa cells. Jurkat T cells were treated similarly except apoptosis was additionally induced by incubation in the presence of 4% EtOH. Epithelial cells fused with the highest efficiencies of lipid mixing, when pretreated with staurosporine. Jurkat T cells were less susceptible to fusion, but they also displayed an increase in fusion efficiency after the induction of apoptosis. Alternatively, we treated the cells with metabolic inhibitors causing ATP-depletion in order to inactivate aminophospholipid translocase. After ATP-depletion, HeLa and Jurkat T cells fused with DOTAP liposomes with higher efficiencies than control cells. Our conclusion is that the lipid asymmetry of natural membranes may limit fusion with cationic liposomes.

Cystocyte and lymphocyte derived fusomes/spectrosomes: analogies and differences: a mini-review.

Structures analogous to Drosophila spectrosomes were found in mammalian lymphocytes. Repasky and colleagues discovered an intracellular spectrin-rich structure in lymphoid cells, which had far-reaching parallels with the fusome/spectrosome of D. melanogaster germ cells. This fact implies that spectrosomes may be characteristic not only of insect germ cells, but also that an analogous structure may play an important role in other cell types. The term "spectrosome" was first used by Lin and Spradling in 1995 to describe a large sphere of fusomal material in D. melanogaster germline stem cells and their differentiated daughter cells - cytoblasts. In the D. melanogaster ovary, membrane skeletal proteins such as ankyrin, alpha/beta spectrin as well as adducin-like Hts protein(s) were found in this specific organelle - spectrosome/fusome. These orgalelles are involved in the creation of mitotic spindles and D. melanogaster cyst formation and oocyte differentiation, but the role of analogous spectrin-based aggregates found in nucleated cells still remains unclear.