Irreversible incorporation of L-dopa into the C-terminus of α-tubulin inhibits binding of molecular motor KIF5B to microtubules and alters mitochondrial traffic along the axon.

L-dopa is the most effective drug used to date for management of Parkinson's disease symptoms. Unfortunately, long-term administration of L-dopa often results in development of motor disorders, including dyskinesias. Despite extensive research on L-dopa-induced dyskinesia, its pathogenesis remains poorly understood. We demonstrated previously that L-dopa can be post-translationally incorporated into the C-terminus of α-tubulin in living cells. In the present study, we investigated the effect of the presence of L-dopa-tubulin-enriched microtubules on mitochondrial traffic mediated by molecular motor KIF5B. Using biochemical approaches in combination with experiments on neuronal cell lines and mouse hippocampal primary cultures, we demonstrated that L-dopa incorporation into tubulin is irreversible. Transport of mitochondria along the axon was altered after L-dopa treatment of cells. In L-dopa-treated cells, mitochondria had reduced ability to reach the distal segment of the axon, spent more time in pause, and showed reduced velocity of anterograde movement. KIF5B motor, a member of the kinesin family involved in mitochondrial transport in neurons, showed reduced affinity for Dopa-tubulin-containing microtubules. Our findings, taken together, suggest that tyrosination state of tubulin (and microtubules) is altered by L-dopa incorporation into tubulin; the gradual increase in amount of altered microtubules affects microtubule functioning, impairs mitochondrial traffic and distribution, and this could be relevant in Parkinson's disease patients chronically treated with L-dopa.

Extranuclear Structural Components that Mediate Dynamic Chromosome Movements in Yeast Meiosis.

Telomere-led rapid chromosome movements or rapid prophase movements direct fundamental meiotic processes required for successful haploidization of the genome. Critical components of the machinery that generates rapid prophase movements are unknown, and the mechanism underlying rapid prophase movements remains poorly understood. We identified S. cerevisiae Mps2 as the outer nuclear membrane protein that connects the LINC complex with the cytoskeleton. We also demonstrate that the motor Myo2 works together with Mps2 to couple the telomeres to the actin cytoskeleton. Further, we show that Csm4 interacts with Mps2 and is required for perinuclear localization of Myo2, implicating Csm4 as a regulator of the Mps2-Myo2 interaction. We propose a model in which the newly identified functions of Mps2 and Myo2 cooperate with Csm4 to drive chromosome movements in meiotic prophase by coupling telomeres to the actin cytoskeleton.

Post-translational incorporation of 3,4-dihydroxyphenylalanine into the C terminus of α-tubulin in living cells.

The C-terminal tyrosine (Tyr) of the α-tubulin chain is subjected to post-translational removal and readdition in a process termed the "detyrosination/tyrosination cycle". We showed in previous studies using soluble rat brain extracts that l-3,4-dihydroxyphenylalanine (l-Dopa) is incorporated into the same site as Tyr. We now demonstrate that l-Dopa incorporation into tubulin also occurs in living cells. We detected such incorporation by determining the "tyrosination state" of tubulin before and after incubation of cells in the presence of l-Dopa. The presence of a tubulin isospecies following l-Dopa incubation that was not recognized by antibodies specific to Tyr- and deTyr-tubulin was presumed to reflect formation of Dopa-tubulin. l-Dopa was identified by HPLC as the C-terminal compound bound to α-tubulin. l-Dopa incorporation into tubulin was observed in Neuro 2A cells and several other cell lines, and was not due to de novo protein biosynthesis. Dopa-tubulin had microtubule-forming capability similar to that of Tyr- and deTyr-tubulin. l-Dopa incorporation into tubulin did not notably alter cell viability, morphology, or proliferation rate. CAD cells (a neuron-like cell line derived from mouse brain) are easily cultured under differentiating and nondifferentiating conditions, and can be treated with l-Dopa. Treatment of CAD cells with l-Dopa and consequent increase in l-Dopa-tubulin resulted in reduction of microtubule dynamics in neurite-like processes.

Post-Translational Incorporation of L-Phenylalanine into the C-Terminus of α-Tubulin as a Possible Cause of Neuronal Dysfunction.

α-Tubulin C-terminus undergoes post-translational, cyclic tyrosination/detyrosination, and L-Phenylalanine (Phe) can be incorporated in place of tyrosine. Using cultured mouse brain-derived cells and an antibody specific to Phe-tubulin, we showed that: (i) Phe incorporation into tubulin is reversible; (ii) such incorporation is not due to de novo synthesis; (iii) the proportion of modified tubulin is significant; (iv) Phe incorporation reduces cell proliferation without affecting cell viability; (v) the rate of neurite retraction declines as level of C-terminal Phe incorporation increases; (vi) this inhibitory effect of Phe on neurite retraction is blocked by the co-presence of tyrosine; (vii) microtubule dynamics is reduced when Phe-tubulin level in cells is high as a result of exogenous Phe addition and returns to normal values when Phe is removed; moreover, microtubule dynamics is also reduced when Phe-tubulin is expressed (plasmid transfection). It is known that Phe levels are greatly elevated in blood of phenylketonuria (PKU) patients. The molecular mechanism underlying the brain dysfunction characteristic of PKU is unknown. Beyond the differences between human and mouse cells, it is conceivable the possibility that Phe incorporation into tubulin is the first event (or among the initial events) in the molecular pathways leading to brain dysfunctions that characterize PKU.

Extrinsic Functions of Lectin Domains in O-N-Acetylgalactosamine Glycan Biosynthesis.

Glycan biosynthesis occurs mainly in Golgi. Molecular organization and functional regulation of this process are not well understood. We evaluated the extrinsic effect of lectin domains (ß-trefoil fold) of polypeptide GalNAc-transferases (ppGalNAc-Ts) on catalytic activity of glycosyltransferases during O-GalNAc glycan biosynthesis. The presence of lectin domain T3lec or T4lec during ppGalNAc-T2 and ppGalNAc-T3 catalytic reaction had a clear inhibitory effect on GalNAc-T activity. Interaction of T3lec or T4lec with ppGalNAc-T2 catalytic domain was not mediated by carbohydrate. T3lec, but not T2lec and T4lec, had a clear activating effect on Drosophila melanogaster core 1 galactosyltransferase enzyme activity and a predominant inhibitory effect on in vivo human core 1 glycan biosynthesis. The regulatory role of the ß-trefoil fold of ppGalNAc-Ts in enzymatic activity of glycosyltransferases involved in the O-glycan biosynthesis pathway, described here for the first time, helps clarify the mechanism of biosynthesis of complex biopolymers (such as glycans) that is not template-driven.

In vivo immunomodulatory effect of the lectin from edible mushroom Agaricus bisporus.

Lectins are glycan-binding proteins that are resistant to digestion in the gastrointestinal tract and enter intact to blood circulation. The aim of this study was to evaluate the influence of edible mushroom Agaricus bisporus lectin (ABL) on innate and adaptive immune responses as well as its effect in two different experimental pathologies that involve the immune system. ABL inhibited in vitro nitric oxide (NO) production by mouse peritoneal macrophages in response to the pro-inflammatory stimuli lipopolysaccharides (LPS). However, it did not modify the activity of arginase, showing that while ABL downregulates M1 activation, it does not affect M2 activation. ABL also inhibited mononuclear cell proliferation in response to mitogen Con A, or in a mixed lymphocyte reaction. During the in vivo studies, oral administration of ABL to BALB/c mice induced a marked inhibition of NO production by peritoneal macrophages after LPS stimuli. The influence of ABL on tumor growth was studied in BALB/c mice receiving daily oral doses of ABL and implanted with CT26 tumor cells. ABL treatment induced significantly higher rate of tumor growth when compared with control mice. On the other hand, oral ABL administration in Wistar rats induced a marked diminution of the incidence of the disease and the severity of the clinical signs of experimental autoimmune encephalomyelitis. We can conclude that ABL has an in vivo immunomodulatory effect reducing the innate and adaptive responses. This food lectin shows potential therapeutic application on control of inflammatory autoimmune pathologies.

Mucosal priming of newborn mice with S. Typhi Ty21a expressing anthrax protective antigen (PA) followed by parenteral PA-boost induces B and T cell-mediated immunity that protects against infection bypassing maternal antibodies.

The currently licensed anthrax vaccine has several limitations and its efficacy has been proven only in adults. Effective immunization of newborns and infants requires adequate stimulation of their immune system, which is competent but not fully activated. We explored the use of the licensed live attenuated S. Typhi vaccine strain Ty21a expressing Bacillus anthracis protective antigen [Ty21a(PA)] followed PA-alum as a strategy for immunizing the pediatric population. Newborn mice primed with a single dose of Ty21a(PA) exhibited high frequencies of mucosal IgA-secreting B cells and IFN-gamma-secreting T cells during the neonatal period, none of which was detected in newborns immunized with a single dose of PA-alum. Priming with Ty21a(PA) followed by PA-boost resulted in high levels of PA-specific IgG, toxin neutralizing and opsonophagocytic antibodies and increased frequency of bone marrow IgG plasma cells and memory B cells compared with repeated immunization with PA-alum alone. Robust B and T cell responses developed even in the presence of maternal antibodies. The prime-boost protected against systemic and respiratory infection. Mucosal priming with a safe and effective S. Typhi-based anthrax vaccine followed by PA-boost could serve as a practical and effective prophylactic approach to prevent anthrax early in life.

Glycan bioengineering in immunogen design for tumor T antigen immunotargeting.

Bioengineering of Galbeta3GalNAcalpha, known as Thomsen-Friedenreich disaccharide (TFD), is studied to promote glycan immunogenicity and immunotargeting to tumor T antigen (Galbeta3GalNAcalpha-O-Ser/Thr). Theoretical studies on disaccharide conformations by energy minimization of structures using MM2 energy function showed that pentalysine (Lys5) linker and benzyl (Bzl) residue enhance TFD rigidity of the glycosidic bond. Antibodies raised against BzlalphaTFD-Lys5 immunogen recognize tumor T antigen. Competitive assays confirm that TFD-related structures are the main glycan epitope. Antibodies produced by glycan bioengineering recognize HT29, T47D, MCF7, and CT26 epithelial tumor cells. Epithelial tumor cell adhesion to T antigen-binding lectins and endothelial cells was lower in the presence of antibodies raised against the engineered immunogen. The immune response directed to the bioengineered glycoconjugate inhibited CT26 tumor cell proliferation and reduced tumor growth in an in vivo mouse model. These results show that TFD bioengineering is a useful immunogenic strategy with potential application in cancer therapy. The same approach can be extended to other glycan immunogens for immunotargeting purposes.

Nitric oxide mediates the suppressive effect of testosterone on cell proliferative response to myelin basic protein.

This study assessed whether the in vitro effect of testosterone on the proliferative response of mononuclear cells to myelin basic protein (MBP) could be mediated by nitric oxide (NO). Testosterone but not cholesterol supplementation specifically suppressed the proliferative response of rat mononuclear cells to MBP and in parallel increased the NO level. NG-monomethyl 1-l-arginine, an inhibitor of NO synthesis, reverted the suppression of the testosterone-induced proliferative response to MBP. These results indicate that changes in the production of NO by testosterone are able to alter the specific T cell proliferation induced by the encephalitogenic MBP and in this way; it could be one of the molecular mechanisms that modulate the development of experimental autoimmune encephalomyelitis (EAE).

Age-related changes in the development of experimental autoimmune encephalomyelitis.

A prominent feature of multiple sclerosis is its high incidence of onset in the third decade of life and its relatively rare onset in persons older than 50 years. In order to study age-related restriction of clinical expression, a comparative biochemical, immunological and histological study was undertaken during development of experimental autoimmune encephalomyelitis (EAE) in young (7 weeks) and middle-aged (15 months) Wistar rats. Young rats showed characteristic clinical signs 12-16 days postinduction, and then they spontaneously recuperated. In middle-aged rats, the incidence of clinical signs was significantly reduced, with a later onset of the disease. Similar biochemical and histological alterations were detected in both age groups, but they were present in a later stage in middle-aged animals. However, cellular and humoral immune responses to myelin basic protein (MBP) were observed 15 days postinduction in all EAE animals. The study of anti-MBP IgG isotype pattern in 7-week-old animals indicated a predominant Th1-type immune response during the acute stage of EAE, with antibodies predominantly recognizing the MBP 96-128 peptide. In contrast, 15-month-old animals showed a less prominent Th1 response, without any epitope dominance. The changes in immune function found in middle-aged animals may account for the different susceptibility and expression of EAE, and may also be relevant to the different clinical expression observed in multiple sclerosis with maturation.

Interaction between gonadal steroids and neuroimmune system in acute experimental autoimmune encephalomyelitis (EAE) in Wistar rats.

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the CNS mediated by autoreactive T lymphocytes directed against myelin antigens. Since neuroendocrine-immune dysfunction appears to contribute to the pathogenesis of autoimmune diseases, the present work was designed to study the effect of changes in the endocrine system on the development of acute EAE and the immune response against myelin basic protein (MBP). Intact and sham males and intact female Wistar rats showed the most severe clinical symptoms (acute period) 12-14 days post-inoculation (dpi). Then, they began gradually to recover, regaining the total ability to walk by 15-17 dpi. Male Wistar rats with altered levels of gonadal hormones by surgical castration showed an onset of the symptoms retarded 2-3 days with respect to the other EAE groups, showing neuropathological symptoms up to 27-28 dpi, and remaining with lower body weight even at 40 dpi. The castrated animals exhibited a specific delay in MBP-stimulated DTH reactivity that correlates with the delay in the onset of the clinical symptoms. Also significant lymphocyte proliferation to MBP was still present at 35 dpi that was absent in the sham group. The distribution of the IgG subclasses indicated that at 35 dpi castrated animals have a higher IgG2b/IgG1 ratio (35.1) in comparison to that presented by sham rats (4.8). Considering that at this time the castrated animals were not completely recuperated, these results could indicate an ongoing inflammatory immune response associated with Th1 activity in these animals. Also castrated animals developed antibodies to a diversity of MBP epitopes in comparison to sham rats, which presented a dominance of antibodies to MBP peptide p96-128. These results indicate that sex hormones levels regulate cell-mediated immunity and the specificity of anti-MBP antibodies related to the induction and development of acute EAE.

Mannan from Aloe saponaria inhibits tumoral cell activation and proliferation.

In this study, we tested the antiproliferative effects of mannan from Aloe saponaria using normal murine (SpMC) and human cells (PBMC) and several tumoral cell lines. Employing flow cytometry, it could be determined that mannan inhibits the proliferative response in normal and tumoral cells. Mannan affects the expression of CD3(+) SpMC indicating that mannan inhibits mainly T lymphocyte proliferative response. Also in SpMC cultured with or without mitogen mannan produces an increase of an activation marker (CD25). On C1498 cell line, mannan reduces CD3 expression and abolishes the CD25 expression. In conclusion, mannan has a dual beneficial effect when applied to normal and tumoral cells at the same time by inhibiting the activation of cancer cells and improving that of normal ones.

Neuronal antigens modulate the immune response associated with experimental autoimmune encephalomyelitis.

Rats primed with bovine myelin (BM) in complete Freunds adjuvant, develop acute experimental autoimmune encephalomyelitis (EAE). We have previously described that intraperitoneal administration prior to the active induction of the disease of a bovine synaptosomal fraction (BSF) and BM were effective ways of suppressing EAE. We found that both treatments diminish the incidence of the disease and reduced biochemical and histological alterations of the central nervous system (CNS). To characterize this suppression process, in this study we examined the antigen-specific immune response in animals protected from EAE. Lymph node mononuclear cells derived from sick EAE rats, as well as from those protected by BM and BSF, showed strong myelin basic protein (MBP) proliferation. Analysis of the humoral response against MBP showed a significant diminution of IgG2b anti-MBP titres in protected BM and BSF rats in contrast to sick EAE rats whose condition could be related to a diminished anti-MBP Th1 response. Finally, cells from rats protected by BSF and BM reduced the incidence of EAE when they were adoptively transferred into animals prior to active induction of the disease. These results suggest that a mechanism based on the generation of regulatory cells and immune deviation could account for the EAE suppression mediated by myelin as well as synaptosomal antigens.