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Exosomes are extracellular membrane vesicles secreted by cells into biological fluids. The membrane of exosomes protects their contents from degradation and contains markers of the cells producing them. Almost all cells of the body produce exosomes, however, tumor cells secrete them more intensively. However, initial stages of the study of exosome functions and identification of tumor protein biomarkers in their composition meet a serious problem of isolating pure, characterized exosome preparations. In this study we have performed quantitative proteomic analysis of human serum exosomes isolated using differential ultracentrifugation, ultracentrifugation in a sucrose cushion, or sedimentation of a serum exosomal fraction by means of the commercial Exosome Isolation Kit (Invitrogen from ThermoFisher Scientific Baltics, UAB, Lithuania). The protein composition of the obtained exosome samples was determined by mass spectrometric methods of selected reactions monitoring (SRM) and shotgun proteomic analysis. The resultant preparations were characterized by the content of the main markers (CD9, CD82, HSPA8, CD63). In the exosomes isolated from serum of healthy volunteers samples by ultracentrifugation in the sucrose cushion, the content of the above mentioned markers was determined as 32.85, 15.59, 6.07 fmol/μg of total protein, respectively. It was shown that the centrifugation method with the sucrose cushion was optimal for the isolation of exosomes. The other methods, including the commercial kit, did not yield positive results. Thus, results of this study have shown that the centrifugation using the sucrose cushion is the most optimal for serum exosome isolation. [ABSTRACT FROM AUTHOR]

Nucleolin (NCL) participates in DNA transcription, ribosomal biogenesis and the regulation of RNA stability. However, the contribution of NCL to tumor development is still not clear. Herein, we found that NCL expression correlated with poor prognosis in lung cancer patients. Overexpressed NCL was predominantly cleaved to C-terminal truncated NCL (TNCL). In lung cancer formation, activation of the epidermal growth factor receptor pathway induced NCL expression, and also the expression of matrix metalloproteinase (MMP) 7, which then cleaved NCL at Asp255 to generate TNCL of 55 kDa. TNCL increased the expression of several oncogenes, including MMP9, anaplastic lymphoma kinase (ALK), HIF1a and CBLB, and decreased the expression of tumor suppressors including BRD4, PCM1, TFG and KLF6 by modulating mRNA stability through binding to the 3'-untranslated regions of their transcripts, thus ultimately enhancing metastasis activity. In conclusion, this study identified a novel role of the cleavage form of NCL generated by MMP7 in stabilizing MMP9 mRNA. We also provide a new insight that MMP7 not only cleaves the extracellular matrix to promote tumor invasion but also cleaves NCL, which augment oncogenesis. Blocking NCL cleavage may provide a useful new strategy for lung cancer therapy. [ABSTRACT FROM AUTHOR]

Purpose To evaluate the effectiveness of adding gait speed to the history of falls in predicting falls among men aged 80 years and older in Taiwan. Methods This prospective cohort study recruited 230 ambulatory men aged 80 years and older in 2012 and followed for 12 months. In addition to demographic characteristics and history of falls, a comprehensive geriatric assessment was performed for all study subjects. Gait speed was obtained by the 6-m walk and three different cut-offs (< 0.5, ≤ 0.8 and < 1.0 m/s) were tested in improving the ability of predicting subsequent falls by using history of falls. Results Among all subjects (mean age: 85.5 ± 4.0 years), 26.1% (60/230) reported falls during follow-up period. Univariate analysis showed that polypharmacy, urinary incontinence, history of falls, pain, poorer baseline physical function, depressive mood, and gait speed < 0.5 m/s were associated with falls. Logistic regression showed that history of falls (OR: 4.255, 95% CI 2.089–8.667; P < 0.001), pain (OR: 2.674, 95% CI 1.332–5.369; P = 0.006), older age (OR: 1.128, 95% CI 1.031–1.234; P = 0.008), and slow gait speed (OR: 2.964, 95% CI 1.394–6.300; P = 0.005) were all independent risk factors for falls. Fast gait speed (defined as ≥ 1 m/s) was a protective factor for falls, even among subjects with history of falls, but slow gait speed (defined as < 0.5 m/s) was an independent risk factor even among subjects without history of falls. Conclusions Combined history of falls and gait speed is a simple and effective tool in risk assessment of falls among older old population. [ABSTRACT FROM AUTHOR]

Summary. Recent studies indicate a possible link between serum cholesterol level, β-amyloid (Aβ) peptide concentrations, and the incidence of Alzheimer's disease (AD). In the present report, the effects of dietary cholesterol on Aβ and apolipoprotein E (APOE) levels in several brain regions were examined using diet-induced hypercholesterolemic rabbits as the animal model. Increased dietary cholesterol levels increased Aβ concentrations in temporal cortex (p = 0.02). A similar trend was observed in the frontal cortex (p = 0.06), yet not in the cerebellum. Interestingly, the regional levels of Aβ in the hypercholesterolemic rabbit paralleled the amyloid pathology observed in AD brain. Elevated APOE levels were also noticed in temporal (p < 0.01) and frontal (p < 0.01) cortices, but not in cerebellum, in the rabbit fed with cholesterol-abundant diet. These results suggest that high serum cholesterol levels could induce the elevation of brain APOE, which may play a role in aggravating the Aβ accumulation. [ABSTRACT FROM AUTHOR]

Summary. Formation of 6-hydroxydopamine (6-OHDA) has been posited in the striatum following methamphetamine treatment and plays a critical role in methamphetamine-induced nigrostriatal dopaminergic toxicity. We used high performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) to determine the formation of 6-OHDA by the treatments of methamphetamine combined with pargyline, a monoamine oxidase inhibitor, and pyrogallol, a catechol-O-methyl-transferase inhibitor, in female C57BL/6J mouse striatum. A substantial amount of 6-OHDA (9.9 ± 0.7 pg/mg wet tissue) was detected in mice treated with pargyline (100 mg/kg) and pyrogallol (25 mg/kg) in combination. Greater striatal 6-OHDA levels were observed in mice treated with combined pargyline, pyrogallol and methamphetamine (50 mg/kg) as compared to mice treated with combined pargyline and pyrogallol. However, mice treated with pargyline and pyragollol in combination followed by one and two doses of methamphetamine exhibited comparable striatal 6-OHDA levels (23.2 ± 4.3, 27.3 ± 1.3 pg/mg wet tissue) in our protocol. We conclude that blockade of the primary metabolic pathways of dopamine by inhibiting both monoamine oxidase and catechol-O-methyl-transferase activities is sufficient to induce 6-OHDA formation in the striatum. Acute 6-OHDA accumulation in the striatum can be potentiated by methamphetamine, a potent dopamine releaser, administration following such metabolic inhibitions. [ABSTRACT FROM AUTHOR]

Summary. Intra-striatal infusion of a high dose (100 μg/3 μl) of methamphetamine produced long-lasting depletions of striatal dopamine and serotonin in both male and female mice. Male mice exhibited a greater depletion of striatal dopamine and serotonin than female mice. A similar trend of sexual differences was observed when 4 cumulative doses of methamphetamine were administered systemically. Thus, the sexual differences in methamphetamine-induced neurotoxicity in the striatum are probably not due to their differences in peripheral metabolism of methamphetamine. Moreover, ovariectomized (OVX) mice supplemented with 3 daily doses of estradiol benzoate (EB) at high or physiological levels, 3 daily doses of progesterone (P), and 2 doses of EB followed by 1 dose of P all demonstrated higher striatal dopamine levels following methamphetamine treatment as compared to vehicle-supplemented controls. The OVX mice pretreated with 3 daily doses of P exhibited the highest striatal serotonin levels after methamphetamine administration of all groups. In conclusion, sexual differences observed in methamphetamine-induced striatal neurotoxicity may be modulated by ovarian hormones. [ABSTRACT FROM AUTHOR]

Summary. Four consecutive doses (10 mg/kg) of methamphetamine, s.c., produced a substantial striatal dopamine depletion in both sexes of BALB/c and C57BL/6J mice. Male C57BL/6J mice exhibited greater dopamine depletions in the striatum compared to female C57BL/6J mice. In contrast, male and female BALB/c mice demonstrated an equivalent magnitude of striatal dopamine depletion. Regardless of sex, C57BL/6J mice demonstrated approximately 1.4 to 2.2 times greater dopamine depletions in the striatum compared to BALB/c mice. Moreover, methamphetamine caused 4 times greater serotonin depletions in male as opposed to female BALB/c mice while sparing either sex of the C57BL/6J mice. Furthermore, female mice of both strains appeared to have the greatest basal dopamine levels during proestrus and the lowest basal dopamine levels during diestrus. Likewise, female mice of both strains exhibited the lowest dopamine depletions in the striatum when the dosing regimen of methamphetamine started at proestrus whereas the greatest dopamine depletions in the striatum occurred when the regimen started during diestrus. These results suggest that sex hormones and other modulating factors may play a role in methamphetamine-induced dopamine and serotonin neurotoxicity. [ABSTRACT FROM AUTHOR]