Pao-Chi Liao

Sexual differences and estrous cycle in methamphetamine-induced dopamine and serotonin depletions in the striatum of mice

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.n

Micro devices integrated with microchannels and electrospray nozzles using PDMS casting techniques

Abstract The design, fabrication, and analytical utility of a polydimethylsiloxane (PDMS)-based microfluidic device for electrospray ionization mass spectrometry (ESI-MS) are described. The microdevice is composed of a one-dimensional (1D), or three-dimensional (3D) channel integrated with sample reservoirs, built-in electrodes and silica capillaries as electrospray nozzles. Several innovative fabrication features have been reported in the present study. First, there is no dead-volume region in the connection of the microchannels and capillary nozzles using PDMS casting techniques. Furthermore, there is no bonding process required to form a sealed microchannel, resulting in a simpler fabrication process and providing higher mechanical strength for high-pressure applications. Another advantage of the developed method is the feasibility to fabricate genuine 3D channels integrated with electrospray nozzles such that chip size can be minimized. Electrical contact to apply required high drive voltage for generation of the electrosprays can be also integrated on the microfluidic chip. The micro devices could be mass-produced at low costs and used as a disposable device to generate ESI-MS signals for protein identification from low amounts of protein samples. Compared with commercially available nanospray capillary tips, the microfluidic module gives comparable signal quality for ESI-MS and also offers advantages in convenience and easiness in operation, permitting repeated usages and disposability.

Estrogen and progesterone distinctively modulate methamphetamine-induced dopamine and serotonin depletions in C57BL/6J mice

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.

A disposable poly(methylmethacrylate)-based microfluidic module for protein identification by nanoelectrospray ionization-tandem mass spectrometry.

The design, fabrication, and analytical use of a poly(methylmethacrylate) (PMMA)‐based microfluidic module for nanoelectrospray ionization‐tandem mass spectrometry (nano‐ESI‐MS/MS) were described. The microfluidic module can be mass‐produced at low costs and used as a disposable device to generate nano‐ESI‐MS/MS signals for protein identification from low amounts of protein samples. Compared with commercially available nanospray capillary tips, the module gave comparable signal quality and also offered advantages in convenience and easiness of operation, permitting repeated usage, and disposability.

Striatal formation of 6-hydroxydopamine in mice treated with pargyline, pyrogallol and methamphetamine.

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 ± 0.7u2009pg/mg wet tissue) was detected in mice treated with pargyline (100u2009mg/kg) and pyrogallol (25u2009mg/kg) in combination. Greater striatal 6-OHDA levels were observed in mice treated with combined pargyline, pyrogallol and methamphetamine (50u2009mg/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 ± 1.3u2009pg/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.

Biochemical characterization of sialoprotein "anti-agglutinin" purified from boar epididymal and seminal plasma.

Sialoprotein “anti‐agglutinin,” previously shown to inhibit sperm head‐to‐head agglutination, is found in both boar epididymal and seminal plasma. The present report characterizes anti‐agglutinin by mass spectrometry, by N‐terminal amino acid sequence analysis, and by sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis (PAGE) and Western blotting techniques to assess phosphate content of the molecule. Anti‐agglutinin had the SDS‐PAGE mobility of approximately 25 kDa. By electrospray ionization‐mass spectrometry, however, mass spectra of anti‐agglutinin were characterized by two major peaks (19,379–19,382 Da and 19,395–19,397 Da) and several minor peaks. Mass spectrometry of tryptic peptide fragments of deglycosylated anti‐agglutinin and amino acid sequence analysis revealed that the protein has a unique peptide‐mass fingerprinting of fragments (12,668 Da, 5,209 Da, 1,226 Da, and 1,168 Da) and a novel N‐terminal amino acid sequence (KTDDY AISGA KEEEF YDYME ELYAV), respectively. Additionally Western blot techniques, using commercially available monoclonal antibodies, were used to detect presence of phosphothreonine and phosphoserine substituents, but two different monoclonal antibodies did not detect phosphotyrosine. Moreover, treatment with two different alkaline phosphotases converted the molecule, as assessed by SDS‐PAGE and detection by silver stain, from the parent form of about 25 kDa to forms of approximately 19 kDa (similar to that assigned by mass spectrometry) and/or 15 kDa. Original antiserum generated toward, and reacting with native anti‐agglutinin, reacted only with 19 kDa form. These results are consistent with the conclusion that the native anti‐agglutinin may be a novel protein that is phosphorylated at serine and/or threonine residues. Mol. Reprod. Dev. 55:96–103, 2000.

Microfluidic Device with Integrated Protein Digestion, Peptide Separation and Nanospray Interface on Poly (Dimethylsiloxane) PDMS Substrate

Here we present an easy method to fabricate the poly (dimethylsiloxane) (PDMS)-based microfluidic chips with integrated functions for protein identification by tandem mass spectrometry using replica-molding techniques. This microchip has typical electrophoretic microchannels, a pulled nano-electrospray ionization (ESI) capillary tip at the outlet and a cartridge packed with the trypsin-immobilized beads attached to sample reservoir of the device. Moreover, a novel frit made-up by photopolymer technique was fabricated near the front of the separation channel, where the C18 beads were packed for de-salting. Using β-casein as a test protein, the MS spectra acquired from different migration times indicate that the digested fragments were separated. Moreover, some signature peptides of beta-casein were clearly identified by matching their detected molecular weights as well as sequences with those obtained from the database.