Characterisation of Human 5-Hydroxytryptamine2A and 5-Hydroxytryptamine2C Receptors Expressed in the Human Neuroblastoma Cell Line SH-SY5Y: Comparative Stimulation by Hallucinogenic Drugs

Abstract: Stable transfection of the human neuroblastoma cell line SH-SY5Y with the human 5-hydroxytryptamine_2A (5-HT_2A) or 5-HT_2C receptor cDNA produced cell lines demonstrating ligand affinities that correlated closely with those for the corresponding endogenous receptors in human frontal cortex and choroid plexus, respectively. Stimulation of the recombinant receptors by 5-HT induced phosphoinositide hydrolysis with higher potency but lower efficacy at the 5-HT_2C receptor (pEC₅₀ = 7.80 ± 0.06) compared with the 5-HT_2A receptor (pEC₅₀ = 7.30 ± 0.08). Activation of the 5-HT_2A receptor caused a transient fourfold increase in intracellular Ca²⁺ concentration. Whole-cell recordings of cells clamped at ?50 mV demonstrated a small inward current (2 pA) in response to 10 µM 5-HT for both receptors. There were no differences in potency or efficacy of phosphoinositide hydrolysis among four hallucinogenic [d-lysergic acid diethylamide (LSD), 1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane (DOI), 5-methoxy-N,N-dimethyltryptamine, and mescaline] and three nonhallucinogenic drugs (m-chlorophenylpiperazine, quipazine, and ergotamine). Comparison of equipotent doses producing 20% of the maximal response induced by 5-HT revealed selective activation of the 5-HT_2A receptor by LSD and to a lesser degree by DOI, mescaline, and ergotamine. Quipazine and 5-methoxy-N,N-dimethyltryptamine were relatively nonselective, whereas m-chlorophenylpiperazine selectively activated the 5-HT_2C receptor. It is unlikely therefore that hallucinosis is mediated primarily by activity at the 5-HT_2C receptor, whereas activity at the 5-HT_2A receptor may represent an important but not unique mechanism associated with hallucinogenic drug action.     

Prediction of location of active sites in biologically active peptides

In a previous paper we demonstrated that the short-range compact regions in atrial natriuretic factor (-hANF) predicted by the average distance map (ADM) correspond to its active sites [Kikuchi,J. Protein Chem.11, 579–581 (1992)]. In the present paper we apply the same method to other bioactive peptides and peptidic enzyme inhibitors. We again observe that active sites in each peptide are contained in short-range compact regions predicted by the ADM for the peptide. This demonstrates that the ADM method predicts the possible location of active sites in biologically active peptides in general. The possibility of practical application of the present method to rational drug design is also discussed.     

Electrical conductivity dispersion as a probe of membrane modifications in mouse polyomavirus infected cells in culture

In this report we investigate the inhibition of membrane conductivity, due to the murine polyomavirus infection in permissive cells in culture. We define experimental conditions to have reproducible results and demonstrate that the intensity of the effects on the cell membrane, depends upon the virus titer used in the infection. Finally, the virus dependent effects disappear if the infection is performed in the presence of a drug that inhibits polymavirus DNA replication.     

Isozyme analysis of intercontinental disjuncts within Styrax (Styracaceae): implications for the Madrean —Tethyan hypothesis

Various hypotheses have been put forward to explain the presence of sclerophyllous plant disjuncts between western North America and the Mediterranean region. The Madrean–Tethyan hypothesis postulates that the two regions were floristically connected in the Early to Middle Tertiary by way of a low-latitude migration route. Others deny the possibility of such a route, and instead postulate convergence to xerophytic conditions from more widespread mesophytic ancestors, or suggest long-distance dispersal scenarios. One example of a “Madrean–Tethyan link” between the two regions is composed of four species within the genus Styrax: S. officinalis subsp. officinalis from the Mediterranean region, S. officinalis subsp. redivivus and subsp. fulvescens from California, and three closely related species in Texas and northeastern Mexico (S. texanus, S. platanifolius, and S. youngiae). This group was examined with isozymes to assess whether patterns of genetic variation are consistent with those predicted by the Madrean–Tethyan hypothesis. Ten populations from California, six from the Mediterranean region, and three from Texas were sampled. Pairwise comparisons revealed mean genetic identity (I) estimates of 0.581 between Mediterranean and California populations, 0.470 between Mediterranean and Texas populations, and 0.640 between California and Texas populations. Two populations of a species thought by many to be the closest relative of S. officinalis on morphological grounds (S. jaliscanus) exhibited low I (0.299–0.321) relative to all other group comparisons. Intercontinentally disjunct populations of S. officinalis possessed an I value that warrants species status for the Californian and Mediterranean groups. Divergence time estimates between Madrean and Tethyan Styrax range from 5.0 to 13.8 Mya, too recent to be consistent with the Madrean–Tethyan hypothesis. However, alternative explanations for this disjunction are suboptimal in that they require the invocation of either long-distance dispersal, which appears unlikely in this group, or extinction. Nonetheless, the evidence presented here and in other recent studies casts substantial doubt on the Madrean–Tethyan hypothesis as a general explanation for the presence of Madrean and Tethyan taxa similar in overall appearance. More plants with Madrean–Tethyan distributions must be sampled before definitive conclusions regarding this aspect of Madrean and Tethyan vegetation can be reached.     

Evaluation of Drugs for Treating Obesity

Criteria for the evaluation of new drugs to treat obesity are important as guides for designing clinical trials to test these agents. These criteria must be developed in relation to the realities of obesity, which is a chronic disease associated with morbidity and mortality that is increased by visceral fat deposits. The observation that patients regain weight after stopping drug treatment for obesity argues for the proposition that drugs work only when taken and NOT that the drugs are ineffective. The analogy between the development of treatments for obesity to those for the treatment of hypertension is used to highlight potential areas for new developments. Several features of an ideal drug for the treatment of obesity are suggested. Criteria for evaluating new drugs include both primary and secondary endpoints. The primary endpoint for an anti-obesity drug should be weight loss, possibly by category of success. Losses of total body fat or visceral fat might be alternative primary endpoints. Secondary endpoints include reduction in risk factors for associated diseases and improvement in the quality of life. In trials where vigorous placebo designs including highly aggressive behavior modification or very-low-calorie diets were used, it may be difficult or impossible to detect a response to a drug.     

Use of a gel-forming dipeptide derivative as a carrier for antigen presentation

A dipeptide of the formula Fmoc-Leu-Asp and some other related dipeptides were synthesized in solution by standard methods. When such peptides are dissolved in water at concentrations below 1% at 100 °C and cooled below 60 °C they form turbid solutions and eventaully visocelastic gels at lower temperatures. Such gels are thermoreversible and can also be disrupted by mechanical agitation. At a concentration of 2 mg/ml the peptide Fmoc-Leu-Asp forms an aqueous gel at 60 °C with a shear modulus of 80 Pa measured at a frequency of 1 rad/s. Peptide solutions undergo an abrupt increase in light scattering between 1 and 1.5 mg/ml at both 23 and 60 °C. By analogy with previous observations of other systems, this increse appears to be due to the formation of filamentous micelles and the aggregation of filamaents into a three-dimensional network. When low molecular weight adamantanamine derivatives, which are inherently non-antigenci antiviral drugs, were incorporated into the Fmoc-Leu-Asp gel and injected into rabbits, high titre specific antibodies were efficiently produced without the need for additional adjuvant. Both the physical properties of the gel and its effect on the antigenicity of low molecular weight compounds suggest a number of practical applications.     

Dimerization of beta B2-crystallin: the role of the linker peptide and the N- and C-terminal extensions.

beta B2- and gamma B-crystallins of vertebrate eye lens are 2-domain proteins in which each domain consists of 2 Greek key motifs connected by a linker peptide. Although the folding topologies of beta B2- and gamma B-domains are very similar, gamma B-crystallin is always monomeric, whereas beta B2-crystallin associates to homodimers. It has been suggested that the linker or the protruding N- and C-terminal arms of beta B2-crystallin (not present in gamma B) are a necessary requirement for this association. In order to investigate the role of these segments for dimerization, we constructed two beta B2 mutants. In the first mutant, the linker peptide was replaced with the one from gamma B (beta B2 gamma L). In the second mutant, the N- and C-terminal arms of 15- and 12-residues length were deleted (beta B2 delta NC). The beta B2 gamma L mutant is monomeric, whereas the beta B2 delta NC mutant forms dimers and tetramers that cannot be interconverted without denaturation. The spectral properties of the beta B2 mutants, as well as their stabilities against denaturants, resemble those of wild-type beta B2-crystallin, thus indicating that the overall peptide fold of the subunits is not changed significantly. We conclude that the peptide linker in beta B2-crystallin is necessary for dimerization, whereas the N- and C-terminal arms appear to be involved in preventing the formation of higher homo-oligomers.     

Monte Carlo study of the effect of β2-microglobulin on the binding cleft of the HLA-A2 complex

Peptide recognition by class I products of the major histocompatibility complex requires association of the class I heavy chain with β₂-microglobulin. We present results of Monte Carlo simulations of the β-pleated sheet floor of the human class I MHC molecule, HLA-A2, with and without β₂-microglobulin. We find a significant effect of β₂-microglobulin on the side chains of residues near a region that would accommodate the C-terminus of a bound peptide. By modeling simultaneously each loop and its neighboring strand at either end of the class I cleft, we find that β₂-microglobulin restricts the conformational space of residues that are central to binding peptides. The effect is most pronounced for R97 and H114 and somewhat less important for Y99 and Y116, the latter forming strong hydrogen bonds with neighboring residues in the heavy chain itself.     

Herbivorous insect species in the tree canopy of a relict South African forest

Abstract.

• 1 The herbivorous insects on twelve species of evergreen broadleafed trees were repeatedly sampled over a period of 11 months in a small relict forest on the east coast of South Africa. This extraordinarily speciose forest patch has an unusually high proportion of endemic tree species, some of which are extremely rare.
• 2 The insect herbivore fauna (number of species) seems to be markedly depauperate compared to that reported on native, broadleafed trees from other parts of the world. Some possible reasons for this are discussed.
• 3 The total number of herbivorous insect species on each tree species was strongly correlated with the local relative abundance of the host plant species.
• 4 There was no relationship between the total number of insect herbivore species on each tree species and the relative taxonomic isolation of the trees. The proportion of seemingly unique (= specialist) herbivorous insect species (i.e. those that occurred on one tree species only) was greatest on taxonomically isolated trees.
• 5 A fundamental deficiency in the interpretation of the data in this study, and of many other similar studies that report on the number of insect species on plants, is discussed, namely the lack of clarity on the closeness of the association between individual insect herbivore species and their respective host plants.

     

Colour association influences honey bee choice between sucrose concentrations

Certain colours associated with floral food resources are more quickly learned by honey bees (Apis mellifera) than are other colours. But the impact of colour, and other floral cues, on bee choice behaviour has not yet been determined. In these experiments, colour association and sugar concentration of reward were varied to assess how they interact to affect bee choice behaviour. Thirty-five bees were individually given binary choices between blue and yellow artificial flowers that contained either the same rewards or rewards of different sucrose concentrations. Honey bee choice between sucrose concentrations was affected by colour association and this effect was greatest when absolute difference between rewards was the lowest. The honey bee's ability to maximize energetic profitability during foraging is constrained by floral cue effectiveness.