The kinesin of the flagellum attachment zone in Leishmania is required for cell morphogenesis,cell division and virulence in the mammalian host

Leishmania parasites possess a unique and complex cytoskeletal structure termed flagellum attachment zone (FAZ) connecting the base of the flagellum to one side of the flagellar pocket (FP), an invagination of the cell body membrane and the sole site for endocytosis and exocytosis. This structure is involved in FP architecture and cell morphogenesis, but its precise role and molecular composition remain enigmatic. Here, we characterized Leishmania FAZ7, the only known FAZ protein containing a kinesin motor domain, and part of a clade of trypanosomatid-specific kinesins with unknown functions. The two paralogs of FAZ7, FAZ7A and FAZ7B, display different localizations and functions. FAZ7A localizes at the basal body, while FAZ7B localizes at the distal part of the FP, where the FAZ structure is present in Leishmania. While null mutants of FAZ7A displayed normal growth rates, the deletion of FAZ7B impaired cell growth in both promastigotes and amastigotes of Leishmania. The kinesin activity is crucial for its function. Deletion of FAZ7B resulted in altered cell division, cell morphogenesis (including flagellum length), and FP structure and function. Furthermore, knocking out FAZ7B induced a mis-localization of two of the FAZ proteins, and disrupted the molecular organization of the FP collar, affecting the localization of its components. Loss of the kinesin FAZ7B has important consequences in the insect vector and mammalian host by reducing proliferation in the sand fly and pathogenicity in mice. Our findings reveal the pivotal role of the only FAZ kinesin as part of the factors important for a successful life cycle of Leishmania.     

The structural basis of localizing polo-like kinase to the flagellum attachment zone in Trypanosoma brucei

The polo-like kinase in the deep branching eukaryote Trypanosoma brucei (TbPlk) has many unique features. Unlike all the other polo-like kinases known to associate with the nucleus and controlling both mitosis and cytokinesis, TbPlk localizes to the flagellum attachment zone (FAZ) and regulates only cytokinesis in T. brucei. TbPlk was, however, previously found capable of complementing all the multiple Plk (Cdc5) functions in Saccharomyces cerevisiae, indicating that it has acquired all the functions of Cdc5. In the present study, Cdc5 tagged with an enhanced yellow fluorescence protein (EYFP) localized exclusively in the FAZ of T. brucei, suggesting that the unusual localization and limited function of TbPlk are probably attributed to the particular environment in T. brucei cells. Structural basis for the FAZ localization of TbPlk was further investigated with TbPlk and TbPlk mutants tagged with EYFP and expressed in T. brucei. The results indicated that a kinase-inactive mutant N169A and a TbPlk mutant with the entire kinase domain (KD) deleted both localized to the FAZ. Substantial association with FAZ was also maintained when one of the two polo-boxes (PB1 or 2) or the linker region between them was deleted from TbPlk. But a deletion of both polo-boxes led to a complete exclusion of the protein from FAZ. All the deletion mutants retained the kinase activity, further indicating that the TbPlk kinase function does not play a role for FAZ localization. The two polo boxes in TbPlk are most likely instrumental in localizing the protein to FAZ through potential interactions with certain FAZ structural component(s). A putative cryptic bipartite nuclear targeting signal was identified in TbPlk, which was capable of directing TbPlk into the nucleus when either the kinase activity was lost or the PB1 was deleted from the protein.     

Assembly of the Paraflagellar Rod and the Flagellum Attachment Zone Complex During the Trypanosoma brucei Cell Cycle

Trypanosomes possess a single flagellum that is attached to their cell body via the flagellum attachment zone (FAZ). The FAZ is composed of two structures: a cytoplasmic filament complex and four microtubules situated next to it. There is a complex transmembrane crosslinking of this FAZ to the paraflagellar rod (PFR) and axoneme within the flagellum. We have partially purified the FAZ complex and have produced monoclonal antibodies both against the FAZ and the paraflagellar rod. The two antibodies against the FAZ (L3B2 and L6B3) recognise the cytoplasmic filament in immunofluorescence and in immunoelectron microscopy. On western blot, they detect a doublet of high molecular weight (M(r) 200,000). Two anti-PFR antibodies (L13D6 and L8C4) recognise the paraflagellar rod in immunofluorescence, but show a difference on Western blot: L13D6 recognises both major PFR proteins, whereas L8C4 is specific for only one of them. Using these new antibodies we have shown that although the growth of both cytoplasmic FAZ filament and external PFR are related, their growth initiates at different time points during the cell cycle and the two structures elongate at distinct rates.     

Regional localization of [14C]mescaline in rabbit brain after intraventricular administration

Albino rabbits of either sex were anesthetized, and a cannula was implanted permanently into the lateral ventricle. About 1 week later, the distribution of [¹⁴C]mescaline and its deaminated metabolite, [¹⁴C]trimethoxyphenylacetic acid ([¹⁴C]TMPA) in 12 brain regions was examined at 15, 60, and 180 min after the intraventricular injection of [¹⁴C]mescaline (0.5 mol in 0.05 ml saline).¹⁴C-radioactivity was rapidly distributed in all regions, reaching peak levels within 15 min. The spinal cord, superior colliculus, pons, hypothalamus, caudate, medulla oblongata, and inferior colliculus contained 23–57 nmol/g of mescaline; the thalamus, tegmentum, and cerebellum, 12–15 nmol/g; and the cerebrum and hippocampus, less than 10 nmol/g; the levels of [¹⁴C]TMPA ranged from 0.5 to 5 nmol/g. The levels of [¹⁴C]mescaline and of [¹⁴]TMPA in all brain areas were considerably decreased 180 min after its injection. Pretreatment with chlorpromazine (15 mg/kg, i.p., 30 min) lowered [¹⁴C]mescaline concentrations in the hippocampus, caudate, thalamus, and cerebrum and elevated them in the spinal cord, medulla oblongata, pons, and tegmentum; [¹⁴C]TMPA levels as the percentage of total radioactivity were not affected. Pretreatment with iproniazid (150 mg/kg, i.p., 18 h), on the other hand, uniformly reduced the TMPA levels in all brain areas, with the resultant increases in mescaline levels. The CPZ-effect in lowering the mescaline concentrations in the areas belonging to the limbic system may have significance in explaining its antihallucinogenic effect in humans and its ability to block the altered behavior induced by the latter drug in laboratory animals.     

Behavioral disinhibition by mescaline.

Rats were exposed to a CER procedure in which sucrose drinking was suppressed by a tone previously paired with shock. Suppression of drinking during the tone was reduced by mescaline (50 mg/kg) independently of whether training took place under mescaline or placebo. Additional data on the effect of mescaline on sucrose drinking indicated that the result could not be attributed to an increased drive to drink sucrose. It was proposed that mescaline releases behavior from inhibitory control. A number of studies from the literature were cited which supported this hypothesis.     

Mescaline-induced changes of brain-cortex ribosomes. Role of spermidine in counteracting the destabilizing effect of mescaline on brain-cortex ribosomes

1. The effect of spermidine on the mescaline-induced changes of brain-cortex ribosomes was studied by adding spermidine during the treatment of goat brain-cortex slices with mescaline. 2. Mescaline treatment of brain-cortex slices removed a portion of the endogenous spermidine from ribosomes and this removal was significantly prevented when spermidine was present during mescaline treatment. 3. Spermidine present during mescaline treatment of brain-cortex slices counteracted, to some extent, the destabilizing effect of mescaline on ribosomes with respect to heat denaturation. 4. Mescaline treatment of brain-cortex slices made ribosomes more susceptible to breakdown, releasing protein and RNA, and resulting in loss of ribosomal enzymic activities. However, spermidine present during mescaline treatment counteracted moderately the mescaline-induced ribosomal susceptibility to breakdown and ribosomal loss of enzymic activities. 5. Ribosomes of mescaline-treated cortex slices were rapidly degraded by ribonuclease and trypsin. However, if spermidine was present during mescaline treatment of brain-cortex slices the rates of degradation diminished.     

TbSmee1 regulates hook complex morphology and the rate of flagellar pocket uptake in Trypanosoma brucei

Trypanosoma brucei uses multiple mechanisms to evade detection by its insect and mammalian hosts. The flagellar pocket (FP) is the exclusive site of uptake from the environment in trypanosomes and shields receptors from exposure to the host. The FP neck is tightly associated with the flagellum via a series of cytoskeletal structures that include the hook complex (HC) and the centrin arm. These structures are implicated in facilitating macromolecule entry into the FP and nucleating the flagellum attachment zone (FAZ), which adheres the flagellum to the cell surface. TbSmee1 (Tb927.10.8820) is a component of the HC and a putative substrate of polo‐like kinase (TbPLK), which is essential for centrin arm and FAZ duplication. We show that depletion of TbSmee1 in the insect‐resident (procyclic) form of the parasite causes a 40% growth decrease and the appearance of multinucleated cells that result from defective cytokinesis. Cells lacking TbSmee1 contain HCs with aberrant morphology and show delayed uptake of both fluid‐phase and membrane markers. TbPLK localization to the tip of the new FAZ is also blocked. These results argue that TbSmee1 is necessary for maintaining HC morphology, which is important for the parasite's ability to take up molecules from its environment.     

Effect of mescaline on cerebral cholinesterases and on exploratory behavior in rats]

In this study the Authors had the purpose to evaluate the action of a psychotomimetic substance (mescaline) on behavior (exploring behaviour and spontaneous motility) and on cerebral biochemistry (cholinesterase activity) of rat. The mescaline has shown to be active both on behaviour (increasing the spontaneous motility) and on biochemistry (decreasing the total cholinesterase activity). From the examination of the results of this work it could be cautiously assumed the hypothesis that the behavioural effects of mescaline are related (at least partially) to modifications of the cholinergic system at the cerebral level.     

Radioimmunoassays of 3,4,5-trimethoxyphenethylamine (mescaline) and 2,5-dimethoxy-4-methylphenyl-isopropylamine(DOM)

Mescaline (3,4,5-trimethoxyphenethylamine) and N-succinylmescaline were coupled to human serum albumin with carbodiimide. DOM (2,5-dimethoxy-4-methylphenylisopropylamine) was linked to human serum albumin by reaction with glutaraldehyde. These conjugates were used for immunization of rabbits. Derivatives of mescaline [N-(3′,4′,5′-trimethoxyphenethyl)-4-hydroxyphenylacetamide] and of DOM [N-(2′,5′-dimethoxy-4′-methylphenylisopropyl)-4-hydroxyphenylacetamide], which could be iodinated to high specific activity, were synthesized. The antibodies bound specifically to these iodinated compounds. In competition experiments with a mescaline antiserum, 6 pmoles of mescaline inhibited 50%; with a DOM antiserum, 50% inhibition was observed with 118 pmoles of DOM. Thus, 100 pg of mescaline and 2 ng of DOM can be detected. The specificity of both antibodies is such that structurally related molecules, such as p-methoxy-phenethylamine or 3-methoxytyramine, are several orders of magnitude less effective as inhibitors than the parent molecules. With the use of antimescaline, it was determined that mescaline administered intravenously to rabbits disappeared rapidly from the circulation. The acid was identified as the major metabolite in the serum and urine of these animals.     

A repetitive protein essential for the flagellum attachment zone filament structure and function in Trypanosoma brucei

The flagellum is attached along the length of the cell body in the protozoan parasite Trypanosoma brucei and is a defining morphological feature of this parasite. The flagellum attachment zone (FAZ) is a complex structure and has been characterised morphologically as comprising a FAZ filament structure and the specialised microtubule quartet (MtQ) plus the specialised areas of flagellum: plasma membrane attachment. Unfortunately, we have no information as to the molecular identity of the FAZ filament components. Here, by screening an expression library with the monoclonal antibody L3B2 which identifies the FAZ filament we identify a novel repeat containing protein FAZ1. It is kinetoplastid-specific and provides the first molecular component of the FAZ filament. Knockdown of FAZ1 by RNA interference (RNAi) results in the assembly of a compromised FAZ and defects in flagellum attachment and cytokinesis in procyclic trypanosomes. The complexity of FAZ structure and assembly is revealed by the use of other monoclonal antibody markers illustrating that FAZ1 is only one protein of a complex structure. The cytokinesis defects provide further evidence for the role of an attached flagellum in cellular morphogenesis in these trypanosomes.     

The flagellar attachment zone of Trypanosoma cruzi epimastigote forms

The flagellar attachment zone (FAZ) is an adhesion region of Trypanosoma cruzi epimastigote forms where the flagellum emerges from the flagellar pocket and remains attached to the cell body. This region shows a junctional complex which is formed by a linear series of apposed macular structures that are separated by amorphous material and clusters of intramembranous particles. Two protein groups appear to be important in the FAZ region: a membrane glycoprotein of 72kDa and several high molecular weight proteins. To gain a better understanding of the FAZ region, we compared wild-type Y strain T. cruzi epimastigotes with a mutant cell in which the 72-kDa surface glycoprotein (Gp72), involved in cell body-flagellum adhesion, had been deleted by target gene replacement. Using immunofluorescence confocal microscopy and electron microscopy techniques to analyze the FAZ region the results suggest that, in the absence of Gp72, other proteins involved in the formation of FAZ remain concentrated in the flagellar pocket region. The analysis of a 3-D reconstruction model of wild-type epimastigotes showed that the endoplasmic reticulum and mitochondrion are in intimate association with FAZ, in contrast to the null mutant cells where the endoplasmic reticulum was not visualized.     

Estimation of mescaline and pellotine in Lophophora coulter plants (Cactaceae) by means of the oscillographic polarography

Oscillographic polarography has been applied for the mescaline and pellotine estimation. These alkaloids produce in 0.5 N NaOH electrolyte a sharp peak within the cathode region of the oscillogram, each of them showing different potential. It makes possible to estimate them at a concentration of 5.10(-6) g/ml. All the forms of Lophophora williamsii were found to contain mescaline and lower content of pellothine, L. jourdaniana--to have equal content of both alkaloide, L. diffusa and L. fricii--to contain pellotine and only traces of mescaline. Plants grown in the greenhouse accumulated the same amount of alkaloids as native plants. Grafting on roodstock which does not produce essential amount of the alkaloids, does not affect the ability of Lophophora to synthesize mescaline and pellotine.     

SAS-4 Protein in Trypanosoma brucei Controls Life Cycle Transitions by Modulating the Length of the Flagellum Attachment Zone Filament

The evolutionarily conserved centriole/basal body protein SAS-4 regulates centriole duplication in metazoa and basal body duplication in flagellated and ciliated organisms. Here, we report that the SAS-4 homolog in the flagellated protozoan Trypanosoma brucei, TbSAS-4, plays an unusual role in controlling life cycle transitions by regulating the length of the flagellum attachment zone (FAZ) filament, a specialized cytoskeletal structure required for flagellum adhesion and cell morphogenesis. TbSAS-4 is concentrated at the distal tip of the FAZ filament, and depletion of TbSAS-4 in the trypomastigote form disrupts the elongation of the new FAZ filament, generating cells with a shorter FAZ associated with a longer unattached flagellum and repositioned kinetoplast and basal body, reminiscent of epimastigote-like morphology. Further, we show that TbSAS-4 associates with six additional FAZ tip proteins, and depletion of TbSAS-4 disrupts the enrichment of these FAZ tip proteins at the new FAZ tip, suggesting a role of TbSAS-4 in maintaining the integrity of this FAZ tip protein complex. Together, these results uncover a novel function of TbSAS-4 in regulating the length of the FAZ filament to control basal body positioning and life cycle transitions in T. brucei.     

Hallucinogens potentiate responses to serotonin and norepinephrine in the facial motor nucleus

The present investigation was designed to determine the effect of hallucinogens on the facilitating action of serotonin (5-HT) and norepinephrine (NE) in the facial nucleus. Intravenous administration of d-lysergic acid diethylamide (LSD, 5–10 μg/kg), mescaline (0.5–1.0 mg/kg), or psilocin (0.5–1.0 mg/kg) had no effect by themselves on the glutamate-induced excitation of facial motoneurons. In contrast, the facilitation of facial neuron excitation by iontophoretically applied 5-HT and NE was enhanced 6–10 fold by these hallucinogens. The LSD-enhanced responses to 5-HT and NE continued for at least 4 hours after administration of the hallucinogen. Iontophoretic application of LSD or mescaline (low currents) also markedly potentiated the facilitating effect of 5-HT and NE. Higher currents of LSD (15–40 nA) temporarily antagonized the response to 5-HT. The nonhallucinogen ergot derivatives lisuride and methysergide failed to potentiate the facilitating effects of 5-HT or NE. These observations suggest that hallucinogens potentiate the effect of monoamines on facial motoneurons by increasing the sensitivity of 5-HT and NE receptors. A novel mechanism regarding the psychedelic effects of hallucinogens is discussed.     

Zinc concentrations in mouse embryo and maternal plasma

The effect of a single teratogenic dose of the antiepileptic drug valproic acid and its nonteratogenic metabolite, 2-en-valproic acid, on zinc concentrations in mouse plasma, embryo, and decidua on d 9 of gestation was investigated. The substances were injected subcutaneously (sc) as their sodium salts. In this mouse model, valproic acid induced between 20% (400 mg/kg dose) and 60% (600 mg/kg dose) incidence of exencephaly in living fetuses; 2-en-valproic acid was not teratogenic at these dose levels. The zinc concentrations in plasma were significantly increased 1 and 2 h after administration of both substances. The embryonic zinc concentrations were increased 2 and 4 h after application of both substances. The concentrations of zinc in the decidua were not affected. The similarity of effects of valproic acid and its nonteratogenic analog on zinc concentrations in maternal plasma and embryo suggests that the teratogenicity of a single administration of valproic acid in the mouse is not owing to interference with the zinc metabolism in this species.     

Cell crawling assisted by contractile stress induced retraction

Cell locomotion is a result of a series of synchronized chemo-mechanical processes. Crawling-type cell locomotion consists of three steps: protrusion, translocation, and retraction. Previous works have shown that both protrusion and retraction can produce cell movement. For the latter, a cell derives its propulsive force from retraction induced protrusion mechanism, which was experimentally verified by Chen (1979, "Induction of Spreading During Fibroblast Movement," J. Cell Biol., 81, pp. 684-691). In this paper, using finite element method, we take a computational biomimetic approach to study cell crawling assisted by contractile stress induced de-adhesion at the rear of the focal adhesion zone (FAZ). We assume the formation of the FAZ is driven by receptor-ligand bonds and nonspecific interactions. The contractile stress is generated due to the molecular activation of the intracellular actin-myosin machinery. The exerted contractile stress and its time dependency are modeled in a phenomenological manner as a two-spring mechanosensor proposed by Schwarz (2006, "Focal Adhesions as Mechanosensors: The Two-Spring Model," BioSystems, 83(2-3), pp. 225-232). Through coupling the kinetics of receptor-ligand bonds with contractile stress, de-adhesion can be achieved when the stall value of the contractile stress is larger than a critical one. De-adhesion at the rear end of the FAZ causes a redistribution of elastic energy and induces cell locomotion. Parametric studies were conducted to investigate the connection between the cell locomotion speed and stall stress, and receptor-ligand kinetics. Finally, we provide a scaling relationship that can be used to estimate the cell locomotion speed.     

THE EFFECT OF AN ECTOSYLVIAN EPILEPTOGENIC FOCUS INDUCED BY TOPICAL APPLICATION OF MESCALINE ON NITROGENOUS COMPOUNDS IN THE NEOCORTEX OF CAT

An cctosylvian spiking focus, induced by topical application of mescaline, brought about changes in nitrogenous compounds not only in the focus but throughout the cerebral cortex. Prior to the onset of the spiking focus, 2 min after mescaline application, ammonia levels increased significantly in all neocortical areas, especially at the focus and homolateral marginal gyrus; free amide nitrogen decreased and non-protein nitrogen increased throughout the cortex. During the development of the focus–at 30 min–the ammonia level still remained high, with a tendency to decrease, and free amide nitrogen was decreased in all neocortical areas. A decrease in‘protein nitrogen’in marginal gyri, on either side and in lipid nitrogen in the epileptical focus as well as in both marginal gyri was also noted.     

KMP-11, a basal body and flagellar protein, is required for cell division in Trypanosoma brucei

Kinetoplastid membrane protein 11 (KMP-11) has been identified as a flagellar protein and is conserved among kinetoplastid parasites, but its potential function remains unknown. In a recent study, we identified KMP-11 as a microtubule-bound protein localizing to the flagellum as well as the basal body in both procyclic and bloodstream forms of Trypanosoma brucei (Z. Li, J. H. Lee, F. Chu, A. L. Burlingame, A. Gunzl, and C. C. Wang, PLoS One 3:e2354, 2008). Silencing of KMP-11 by RNA interference inhibited basal body segregation and cytokinesis in both forms and resulted in multiple nuclei of various sizes, indicating a continuous, albeit somewhat defective, nuclear division while cell division was blocked. KMP-11 knockdown in the procyclic form led to severely compromised formation of the new flagellum attachment zone (FAZ) and detachment of the newly synthesized flagellum. However, a similar phenotype was not observed in the bloodstream form depleted of KMP-11. Thus, KMP-11 is a flagellar protein playing critical roles in regulating cytokinesis in both forms of the trypanosomes. Its distinct roles in regulating FAZ formation in the two forms may provide a clue to the different mechanisms of cytokinetic initiation in procyclic and bloodstream trypanosomes.     

Revue des psychodysleptiques: I. Classification des psychodysleptiques

The discovery of new dysleptic drugs has prompted the present review of these substances which influence neuropsychic activity. It is possible to divide dysleptics into two categories: under the first heading fall mescaline, LSD-25, psilocybine, adrenochrome, bufotenine and dimethyltryptamin; the other group includes Ditran, Butoxamine, WH-4849, and AHR-379. The place of Sernyl could not be ascertained and it might well constitute a class by itself. The mescaline type of reaction is mainly characterized by the induction of perceptual distortions without alteration of the state of consciousness, whereas the Ditran type reaction is one of confusion with postexperimental amnesia. Electroencephalographic recordings support this classification: lowering of amplitude, acceleration of rhythm and desynchronization were noted with the first group; and slowing of rhythm and slow waves similar to those seen during the onset of sleep, with the other group.     

Non-enzymic incorporation of amines into proteolipid protein

—Proteolipid protein binds biogenic amines irreversibly. The bound amines can be recovered as the original compounds and as complexes with polypeptide after proteolysis or acid hydrolysis. The properties of the protein-bound tryptamine are consistent with the concept that the amine is covalently linked by its primary amine group. Mescaline, 3,4-dihydroxyphenylethylamine, 5-hydroxytryptamine (5-HT) and histamine also bind irreversibly to proteolipid protein. 5-Hydroxytryptamine and mescaline bind similarly to other proteins (casein, ovalbumin, serum albumin and a serum protein mixture), but proteolipid binds the amines to a greater extent.