Antibacterial, antibiofilm, and antivirulence potential of the main diterpenes from Copaifera spp. oleoresins against multidrug-resistant bacteria.

To evaluate the antibacterial, antibiofilm and antivirulence potential of the main diterpenes from Copaifera spp. oleoresins against multidrug-resistant (MDR) bacteria. Antimicrobial assays included determination of the Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC), Minimum Inhibitory Concentration of Biofilm (MICB50), as well as synergistic and antivirulence assays for eight diterpenes against MDR. The tests revealed that two diterpenes (named 1 and 5) showed the best results, with MIC and MBC between 12.5 and 50 µg/mL against most MDR bacteria. These diterpenes exhibited promising MICB50 in concentration between 3.12-25 µg/mL but showed no synergistic antimicrobial activity. In the assessment of antivirulence activity, diterpenes 1 and 5 inhibited only one of the virulence factors evaluated (Dnase) produced by some strains of S. aureus at subinhibitory concentration (6.25 µg/mL). Results obtained indicated that diterpenes isolated from Copaifera oleoresin plays an important part in the search of new antibacterial and antibiofilm agents that can act against MDR bacteria.

Interstitial lung disease in patients enrolled in early-phase clinical trials: the ILDE study.

BACKGROUND: Interstitial lung disease (ILD) encompasses a heterogeneous group of disorders sharing pathophysiological inflammatory mechanisms, leading to parenchymal distortions. The prevalence of ILD with new cancer drugs is underreported: the identification of potential determinants is priority. MATERIALS AND METHODS: ILDE is a retrospective study aimed at describing the clinical course and potential determinants of ILD in patients receiving experimental treatments. RESULTS: We identified 226 eligible patients, of whom 5.3% (n = 12) had ILD. In five patients, the diagnosis was radiological, while seven patients had initial cough, dyspnea, fatigue or fever. ILD was graded as grade 1 (G1) in four, G2 in five and G3 in three patients. The first occurrence of ILD resolved completely in 50% of patients (n = 6/12). No patient had fatal ILD. Eight patients (66.7%) resumed the treatment after the first episode of ILD, while four patients (33.3%) had to discontinue the therapy. Five out of six patients had resolved the first ILD episode and then resumed treatment, experiencing a second ILD episode (n = 5/6; 83.3%). The second ILD event was G1 in three patients and G2 in two patients, resulting in three patients who eventually discontinued the treatment (n = 3/5; 60%). Correlation analysis showed a higher risk of ILD in older patients (P = 0.051), those who had received previous chest radiation therapy (P = 0.047) or those receiving antibody-drug conjugates (P = 0.006). In a survival analysis adjusted for immortal time bias, ILD was not independently prognostic (hazard ratio 0.50, 95% confidence interval 0.23-1.09, P = 0.082). CONCLUSIONS: In ILDE, patients experiencing ILD had generally good outcomes, and many could resume the cancer treatments. Implementing best practices to prompt diagnosis and management of ILD is critical to treat a potentially severe adverse effect of new drugs, while not affecting patients' outcomes. Research efforts to identify risk factors is warranted, to implement risk-based monitoring schedules and develop ad hoc strategies to improve the cure rates of ILD.

Subcortical serotonin 5HT2c receptor-containing neurons sex-specifically regulate binge-like alcohol consumption, social, and arousal behaviors in mice.

Binge alcohol consumption induces discrete social and arousal disturbances in human populations that promote increased drinking and accelerate the progression of Alcohol Use Disorder. Here, we show in a mouse model that binge alcohol consumption disrupts social recognition in females and potentiates sensorimotor arousal in males. These negative behavioral outcomes were associated with sex-specific adaptations in serotonergic signaling systems within the lateral habenula (LHb) and the bed nucleus of the stria terminalis (BNST), particularly those related to the receptor 5HT2c. While both BNST and LHb neurons expressing this receptor display potentiated activation following binge alcohol consumption, the primary causal mechanism underlying the effects of alcohol on social and arousal behaviors appears to be excessive activation of LHb5HT2c neurons. These findings may have valuable implications for the development of sex-specific treatments for mood and alcohol use disorders targeting the brain's serotonin system.

TAAC - TMS Adaptable Auditory Control: A universal tool to mask TMS clicks.

BACKGROUND: Coupling transcranial magnetic stimulation with electroencephalography (TMS-EEG) allows recording the EEG response to a direct, non-invasive cortical perturbation. However, obtaining a genuine TMS-evoked EEG potential requires controlling for several confounds, among which a main source is represented by the auditory evoked potentials (AEPs) associated to the TMS discharge noise (TMS click). This contaminating factor can be in principle prevented by playing a masking noise through earphones. NEW METHOD: Here we release TMS Adaptable Auditory Control (TAAC), a highly flexible, open-source, Matlab®-based interface that generates in real-time customized masking noises. TAAC creates noises starting from the stimulator-specific TMS click and tailors them to fit the individual, subject-specific click perception by mixing and manipulating the standard noises in both time and frequency domains. RESULTS: We showed that TAAC allows us to provide standard as well as customized noises able to effectively and safely mask the TMS click. COMPARISON WITH EXISTING METHODS: Here, we showcased two customized noises by comparing them to two standard noises previously used in the TMS literature (i.e., a white noise and a noise generated from the stimulator-specific TMS click only). For each, we quantified the Sound Pressure Level (SPL; measured by a Head and Torso Simulator - HATS) required to mask the TMS click in a population of 20 healthy subjects. Both customized noises were effective at safe (according to OSHA and NIOSH safety guidelines) and lower SPLs with respect to standard noises. CONCLUSIONS: At odds with previous methods, TAAC allows creating effective and safe masking noises specifically tailored on each TMS device and subject. The combination of TAAC with tools for the real-time visualization of TEPs can help control the influence of auditory confounds also in non-compliant patients. Finally, TAAC is a highly flexible and open-source tool, so it can be further extended to meet different experimental requirements.

Biosynthesis and characterization of gold nanoparticles using Brazilian red propolis and evaluation of its antimicrobial and anticancer activities.

Gold nanoparticles (AuNPs) are highlighted due to their low toxicity, compatibility with the human body, high surface area to volume ratio, and surfaces that can be easily modified with ligands. Biosynthesis of AuNPs using plant extract is considered a simple, low-cost, and eco-friendly approach. Brazilian Red Propolis (BRP), a product of bees, exhibits anti-inflammatory, anti-tumor, antioxidant, and antimicrobial activities. Here, we described the biosynthesis of AuNPs using BRP extract (AuNPextract) and its fractions (AuNPhexane, AuNPdichloromethane, AuNPethyl acetate) and evaluated their structural properties and their potential against microorganisms and cancer cells. AuNPs showed a surface plasmon resonance (SPR) band at 535 nm. The sizes and morphologies were influenced by the BRP sample used in the reaction. FTIR and TGA revealed the involvement of bioactive compounds from BRP extract or its fractions in the synthesis and stabilization of AuNPs. AuNPdichloromethane and AuNPhexane exhibited antimicrobial activities against all strains tested, showing their efficacy as antimicrobial agents to treat infectious diseases. AuNPs showed dose-dependent cytotoxic activity both in T24 and PC-3 cells. AuNPdichloromethane and AuNPextract exhibited the highest in vitro cytotoxic effect. Also, the cytotoxicity of biogenic nanoparticles was induced by mechanisms associated with apoptosis. The results highlight a potential low-cost green method using Brazilian red propolis to synthesize AuNPs, which demonstrated significant biological properties.

Production and purification of higher molecular weight chondroitin by metabolically engineered Escherichia coli K4 strains.

The capsular polysaccharide obtained from Escherichia coli K4 is a glycosaminoglycan-like molecule, similar to chondroitin sulphate, that has established applications in the biomedical field. Recent efforts focused on the development of strategies to increase K4 polysaccharide fermentation titers up to technologically attractive levels, but an aspect that has not been investigated so far, is how changes in the molecular machinery that produces this biopolymer affect its molecular weight. In this work, we took advantage of recombinant E. coli K4 strains that overproduce capsular polysaccharide, to study whether the inferred pathway modifications also influenced the size of the produced polymer. Fed-batch fermentations were performed up to the 22 L scale, in potentially industrially applicable conditions, and a purification protocol that allows in particular the recovery of high molecular weight unsulphated chondroitin, was developed next. This approach allowed to determine the molecular weight of the purified polysaccharide, demonstrating that kfoF overexpression increased polymer size up to 133 kDa. Higher polysaccharide titers and size were also correlated to increased concentrations of UDP-GlcA and decreased concentrations of UDP-GalNAc during growth. These results are interesting also in view of novel potential applications of higher molecular weight chondroitin and chondroitin sulphate in the biomedical field.

Molecular, Morphological, and Functional Characterization of Corticotropin-Releasing Factor Receptor 1-Expressing Neurons in the Central Nucleus of the Amygdala.

The central nucleus of the amygdala (CeA) is a brain region implicated in anxiety, stress-related disorders and the reinforcing effects of drugs of abuse. Corticotropin-releasing factor (CRF, Crh) acting at cognate type 1 receptors (CRF1, Crhr1) modulates inhibitory and excitatory synaptic transmission in the CeA. Here, we used CRF1:GFP reporter mice to characterize the morphological, neurochemical and electrophysiological properties of CRF1-expressing (CRF1+) and CRF1-non-expressing (CRF1-) neurons in the CeA. We assessed these two neuronal populations for distinctions in the expression of GABAergic subpopulation markers and neuropeptides, dendritic spine density and morphology, and excitatory transmission. We observed that CeA CRF1+ neurons are GABAergic but do not segregate with calbindin (CB), calretinin (CR), parvalbumin (PV), or protein kinase C-δ (PKCδ). Among the neuropeptides analyzed, Penk and Sst had the highest percentage of co-expression with Crhr1 in both the medial and lateral CeA subdivisions. Additionally, CeA CRF1+ neurons had a lower density of dendritic spines, which was offset by a higher proportion of mature spines compared to neighboring CRF1- neurons. Accordingly, there was no difference in basal spontaneous glutamatergic transmission between the two populations. Application of CRF increased overall vesicular glutamate release onto both CRF1+ and CRF1- neurons and does not affect amplitude or kinetics of EPSCs in either population. These novel data highlight important differences in the neurochemical make-up and morphology of CRF1+ compared to CRF1- neurons, which may have important implications for the transduction of CRF signaling in the CeA.

The decrease of NAD(P)H has a prominent role in dopamine toxicity.

We characterized dopamine toxicity in human neuroblastoma SH-SY5Y cells as a direct effect of dopamine on cell reductive power, measured as NADH and NADPH cell content. In cell incubations with 100 or 500 microM dopamine, the accumulation of dopamine inside the cell reached a maximum after 6 h. The decrease in cell viability was 40% and 75%, respectively, after 24 h, and was not altered by MAO inhibition with tranylcypromine. Dopamine was metabolized to DOPAC by mitochondrial MAO and, at 500 microM concentration, significantly reduced mitochondrial potential and oxygen consumption. This DA concentration caused only a slight increase in cell peroxidation in the absence of Fe(III), but a dramatic decrease in NADH and NADPH cell content and a concomitant decrease in total cell NAD(P)H/NAD(P)+ and GSH/GSSG and in mitochondrial NADH/NAD+ ratios. Dopaminechrome, a product of dopamine oxidation, was found to be a MAO-A inhibitor and a strong oxidizer of NADH and NADPH in a cell-free system. We conclude that dopamine may affect NADH and NADPH oxidation directly. When the intracellular concentrations of NAD(P)H and oxidized dopamine are similar, NAD(P)H triggers a redox cycle with dopamine that leads to its own consumption. The time-course of NADH and NADPH oxidation by dopamine was assessed in cell-free assays: NAD(P)H concentration decreased at the same time as dopamine oxidation advanced. The break in cell redox equilibrium, not excluding the involvement of free oxygen radicals, could be sufficient to explain the toxicity of dopamine in dopaminergic neurons.

Lysine-specific demethylase LSD1 regulates autophagy in neuroblastoma through SESN2-dependent pathway.

Autophagy is a physiological process, important for recycling of macromolecules and maintenance of cellular homeostasis. Defective autophagy is associated with tumorigenesis and has a causative role in chemotherapy resistance in leukemia and in solid cancers. Here, we report that autophagy is regulated by the lysine-specific demethylase LSD1/KDM1A, an epigenetic marker whose overexpression is a feature of malignant neoplasia with an instrumental role in cancer development. In the present study, we determine that two different LSD1 inhibitors (TCP and SP2509) as well as selective ablation of LSD1 expression promote autophagy in neuroblastoma cells. At a mechanistic level, we show that LSD1 binds to the promoter region of Sestrin2 (SESN2), a critical regulator of mTORC1 activity. Pharmacological inhibition of LSD1 triggers SESN2 expression that hampers mTORC1 activity, leading to enhanced autophagy. SESN2 overexpression suffices to promote autophagy in neuroblastoma cells, while loss of SESN2 expression reduces autophagy induced by LSD1 inhibition. Our findings elucidate a mechanism whereby LSD1 controls autophagy in neuroblastoma cells through SESN2 transcription regulation, and we suggest that pharmacological targeting of LSD1 may have effective therapeutic relevance in the control of autophagy in neuroblastoma.

Defective and enhanced postreplication repair in classical and variant xeroderma pigmentosum cells treated with N-acetoxy-2-acetylaminofluorene.

Xeroderma pigmentosum (XP) cells proficient in the excision repair of pyrimidine dimers (XP variants) were also found to be proficient in the excision repair of N-2-acetoxyacetylaminofluorene (AAAF)-induced lesions in their DNA, as assayed by the photolysis of 5-bromodeoxyuridine incorporated during repair. However, the time in which the small segments of newly synthesized DNA, made immediately after treatment of cells with AAAF, were joined together to form DNA of parental size by a process called postreplication repair was long in the XP variant and classical cells. Although increasing doses of AAAF increased the time for making daughter DNA of parental size for variant and classical XP cells, AAAF did not appear to affect this process in normal human cells. Treatment of variant and classical XP cells with a relatively small dose (2.5 micron) of AAAF or 2.5 J/sq m of UV radiation several hr before a 2- to 3-fold-larger dose decreased the time for the pulse-labeled DNA to appear as parental size.

DNA repair following ultraviolet and N-ethyl-N-nitrosourea treatment of cells cultured from human fetal brain, intestine, kidney, liver, and skin.

DNA excision repair was measured in cell cultures derived from human fetal brain, intestine, kidney, liver, and skin following ultraviolet (UV) irradiation and N-ethyl-N-nitrosourea (ENU) treatment. Cells in early passages were exposed to 5 or 10 J of UV radiation per sq m or to 25 microM to 3.5 mM ENU. DNA excision repair was determined by (a) scintillation counting and autoradiography to measure unscheduled DNA synthesis (UDS) and (b) the UV-endonuclease-sensitive site assay to measure pyrimidine dimers directly. The level of UDS following treatment of these cell cultures with UV was both time and dose dependent. UDS also increased with increasing doses of ENU up to 350 microM but decreased at doses greater than 500 microM. Cells derived from human fetal brain, kidney, and liver appeared to exhibit lower (50 to 80%) levels of UDS following UV irradiation or ENU treatment than did cells cultured from human fetal skin or intestine. The loss of UV-endonuclease-sensitive sites assayed in skin, liver, and kidney cells over a 24-hr period confirmed the differences observed by UDS in these cells. Skin cells removed 50% of the initial pyrimidine dimers from their DNA within an 8-hr period and 65 to 86% in 24 hr. Kidney and liver cells, on the other hand, removed only 28 and 32% of the initial dimers, respectively, over a 24-hr period. The data suggest differential excision repair responses following UV irradiation and ENU treatment of cells derived from different human fetal organs.

Analysis of alkylated sites at N-3 and N-7 positions of purines as an indicator for chemical carcinogens.

This study reports a rapid assay to distinguish depurination from other forms of alkaline-labile lesions induced in DNA by alkylating agents. Covalently closed circular duplex PM2 DNA was treated with various alkylating agents such as N-methyl-N-nitrosourea, dimethyl sulfate, methyl methanesulfonate, N-ethyl-N-nitrosourea, diethyl sulfate, and ethyl methanesulfonate at pH 6.5. Apurinic sites and subsequent strand breaks were introduced by the hydrolysis of the alkylated purines under nondenaturing conditions by heating alkylated DNA at 70 degrees for 1.5 hr with 0.05 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid:KOH (pH 7.4), 0.1 M KCl, 0.01 M MgCl2, 0.0005 M ethylenediaminetetraacetate, 0.05 M glycine, and 0.01 M putrescine. The number of strand breaks produced, representing the alkylated sites at N-3 and N-7 positions of purines, were quantitated by electrophoresis in 1% neutral agarose slab gels. These results were compared with previously reported carcinogenic and mutagenic effects of these compounds, and a correlation between the apurinic sites, the total alkylated sites, and the biological effect of the alkylating agent was determined.

O6-methylguanine-DNA methyltransferase in human fetal tissues: fetal and maternal factors.

O6-Methylguanine methyltransferase (O6-MT) was measured and compared in extracts of 7 human fetal tissues obtained from 21 different fetal specimens as a function of fetal age and race and of maternal smoking and drug usage. Liver exhibited the highest activity followed by kidney, lung, small intestine, large intestine, skin, and brain. Each fetal organ homogenate exhibited a 3- to 5-fold level of interindividual variation of O6-MT. There did not appear to be any significant differences of O6-MT as a function of fetal race and age and in the tissues obtained from mothers who smoked cigarettes during pregnancy. The fetal tissues obtained from an individual using phenobarbital exhibited 4-fold increases in O6-MT activity. The tissues obtained from another individual on kidney dialysis were 2- to 3-fold higher than the normal population. These data suggest a possible enhancement of human fetal O6-MT by certain xenobiotics, with little if any modulation by racial factors and maternal smoking habits.

Growth-promoting effect of 12-O-tetradecanoylphorbol-13-acetate on cultured normal human fetal kidney epithelial cells.

Normal human epithelial cells have been reported to be sensitive to growth inhibition by 12-O-tetradecanoylphorbol-13-acetate in contrast to neoplastic counterparts. Our studies with normal human fetal kidney epithelial cells, however, show that 12-O-tetradecanoylphorbol-13-acetate may increase cell density and promote the growth of these cells at early passages. The result, together with three previous reports showing similar effects in normal human melanocytes, prostatic epithelial cells, and an unidentified cell type in human epidermal cell culture, indicates that human cells may exhibit divergent responses to 12-O-tetradecanoyl-phorbol-13-acetate for induction of cellular differentiation or proliferation.

Contact insensitivity of a subpopulation of normal human fetal kidney epithelial cells and of human carcinoma cell lines.

Early passage normal human fetal kidney epithelial cells were inoculated on top of a confluent monolayer of X-ray lethally irradiated human fibroblasts to determine the colony-forming ability of these epithelial cells. The results indicate that the great majority of the epithelial cells did not have the clonogenic ability on the fibroblast cell mat, although they were capable of colony formation on plastic surface without the cell mat. A small subpopulation of these epithelial cells, however, was able to proliferate on the cell mat. These contact-insensitive fetal epithelial cells were found to be deficient in gap junction-mediated intercellular communication, to contain keratin and gamma-glutamyl transpeptidase but not fibronectin. These contact-insensitive cells appear to have greater proliferative potential than the parental cell population and to exist transiently in early passage but not in late passage culture. The ability of proliferation on cell mat was found to be shared by 22 different human carcinoma cell lines that were tested. This unique clonogenic ability of normal contact-insensitive and human carcinoma cells on the cell mat could provide a selection method for presumptive normal stem and tumor cells and for an assay for screening potential antitumor drugs and assessing the efficacy of chemotherapeutic drugs against a given tumor.

The ω-SQUIPT as a tool to phase-engineer Josephson topological materials.

Multi-terminal superconducting Josephson junctions based on the proximity effect offer the opportunity to tailor non-trivial quantum states in nanoscale weak links. These structures can realize exotic topologies in several dimensions, for example, artificial topological superconductors that are able to support Majorana bound states, and pave the way to emerging quantum technologies and future quantum information schemes. Here we report the realization of a three-terminal Josephson interferometer based on a proximized nanosized weak link. Our tunnelling spectroscopy measurements reveal transitions between gapped (that is, insulating) and gapless (conducting) states that are controlled by the phase configuration of the three superconducting leads connected to the junction. We demonstrate the topological nature of these transitions: a gapless state necessarily occurs between two gapped states of different topological indices, in much the same way that the interface between two insulators of different topologies is necessarily conducting. The topological numbers that characterize such gapped states are given by superconducting phase windings over the two loops that form the Josephson interferometer. As these gapped states cannot be transformed to one another continuously without passing through a gapless condition, they are topologically protected. The same behaviour is found for all of the points of the weak link, confirming that this topology is a non-local property. Our observation of the gapless state is pivotal for enabling phase engineering of different and more sophisticated artificial topological materials.

A sequence specific endonuclease from Micrococcus radiodurans.

A new sequence specific endonuclease, Mra I has been purified from Micrococcus radiodurans. This enzyme cleaves bacteriophage lambda DNA at three sites, adenovirus type 2 DNA at more than 12 sites and has a unique site on phi X174 DNA. It has no sites on SV40, PM2 and pBR322 DNA. The three sites on phage lambda DNA are different from those cleaved by Sma I, Xma I and Xor II. The sites of cleavage are located at 0.424, 0.447 and 0.834 fractional lengths on the physical map of lambda DNA. Mra I is shown to be an isoschizomer of Sac II and Sst II recognizing the palindromic nucleotide sequence '5-CCGC reduced GG-3'. The enzyme shows an absolute requirement of Mg2+, but is active in the absence of added 2-mercaptoethanol. The enzyme shows activity at a broad range of temperature and pH with an optimum at 45 degrees C and pH 7.0. Mra I represents the first restriction enzyme from a bacterium whose DNA lacks modified methylated bases.

Excision repair of UV-induced pyrimidine dimers in human skin in vivo.

The induction and loss of pyrimidine dimers in human skin in vivo was determined using UV endonuclease, alkaline sucrose sedimentations, and the fluorescent detection of nonradiolabeled DNA. The number of dimers induced following exposure of the skin to radiation emitted from a Burdick UV-800 sunlamp was quantitated by reacting the extracted DNA with Micrococcus luteus endonuclease specific for pyrimidine dimers. Exposure to 15 and 30 seconds of radiation emitted from this lamp produced the formation of 12.8 and 23.6 dimers per 10(8) daltons DNA, respectively. Approximately 50% of the dimers induced were lost 58 min after irradiation. Only a small percentage (less than 10) remained 24 hr postirradiation. These data partially characterize the process by which pyrimidine dimers are excised from human skin DNA in vivo.

DNA repair in skin of lupus erythematosus following in vivo exposure to ultraviolet radiation.

The in vivo repair of pyrimidine dimers induced in the DNA of skin of 9 patients diagnosed as systemic or discoid lupus erythematosus (LE) was measured. A small area of the buttock was exposed to radiation emitted from a Burdick UV-800 sunlamp. The number of pyrimidine dimers was measured by incubating the epidermal skin DNA with UV-specific endonuclease and sedimenting the DNA through alkaline sucrose gradients. The initial number of dimers induced following sunlamp exposure was 7.6 +/- 1.8 per 10(8) daltons DNA. The level of photorepair was measured by illuminating an area of the skin with greater than 450-nm radiation immediately following sunlamp exposure. We found that 56.5 +/- 9.5% of the dimers are photorepaired with 5 min of illumination. Excision repair was measured in an area of the skin covered for 2 and 24 h postirradiation. Approximately 44 and 81% of the dimers induced immediately following sunlamp exposure were removed at these respective times. These observations in LE are similar to those observed in the skin of normal individuals.

Abnormal alpha-synuclein interactions with Rab proteins in alpha-synuclein A30P transgenic mice.

Mutation A30P in the alpha-synuclein gene is a cause of familial Parkinson disease. Transgenic mice expressing wild mouse and mutant human A30P alpha-synuclein, Tg5093 mice (Tg), show a progressive motor disorder characterized by tremor, rigidity, and dystonia, accompanied by accumulation of alpha-synuclein in the soma and neurites and by a conspicuous gliosis beginning in the hippocampal formation at the age of 7 to 8 months and spreading throughout the CNS. Impaired short-term changes in synaptic strength have also been documented in hippocampal slices from Tg mice. Alpha-synuclein aggregates of approximately 34 and 70 kDa, in addition to the band of 17 kDa, corresponding to the molecular weight of alpha-synuclein, were recovered in the PBS-soluble fraction of brain homogenates from Tg mice but not from brain samples from age-matched wildtype littermates. MPTP-treated Tg and wildtype mice produced alpha-synuclein aggregates in the PBS-, deoxycholate-, and SDS-soluble fractions. Aggregates of alpha-synuclein, although with different molecular weights, were also observed in rotenone-treated Tg and wildtype mice. Pull-down studies with members of the Rab protein family have shown that alpha-synuclein from Tg mice interacts with Rab3a, Rab5, and Rab8. This binding is not due to the amount of alpha-synuclein (levels of which are higher in Tg mice) and it is not dependent on the amount of Rab protein used in the assay. Rather, alpha-synuclein interactions with Rab proteins are due to mutant alpha-synuclein as demonstrated in Rab pull-down assays with recombinant of wildtype and mutant A30P human alpha-synuclein. Since Rab3a, Rab5, and Rab8 are important proteins involved in synaptic vesicle trafficking and exocytosis at the synapse, vesicle endocytosis, and trans-Golgi transport, respectively, it can be suggested that these functions are impaired in Tg mice. This rationale is consistent with previous data showing that short-term hippocampal synaptic plasticity is altered and that alpha-synuclein accumulates in the cytoplasm of neurons in Tg mice.