A splice variant of the Drosophila vesicular monoamine transporter contains a conserved trafficking domain and functions in the storage of dopamine, serotonin, and octopamine

Vesicular monoamine transporters (VMATs) mediate the transport of dopamine (DA), serotonin (5HT), and other monoamines into secretory vesicles. The regulation of mammalian VMAT and the related vesicular acetylcholine transporter (VAChT) has been proposed to involve membrane trafficking, but the mechanisms remain unclear. To facilitate a genetic analysis of vesicular transporter function and regulation, we have cloned the Drosophila homolog of the vesicular monoamine transporter (dVMAT). We identify two mRNA splice variants (DVMAT-A and B) that differ at their C-terminus, the domain responsible for endocytosis of mammalian VMAT and VAChT. DVMAT-A contains trafficking motifs conserved in mammals but not C. elegans, and internalization assays indicate that the DVMAT-A C-terminus is involved in endocytosis. DVMAT-B contains a divergent C-terminal domain and is less efficiently internalized from the cell surface. Using in vitro transport assays, we show that DVMAT-A recognizes DA, 5HT, octopamine, tyramine, and histamine as substrates, and similar to mammalian VMAT homologs, is inhibited by the drug reserpine and the environmental toxins 2,2,4,5,6-pentachlorobiphenyl and heptachlor. We have developed a specific antiserum to DVMAT-A, and find that it localizes to dopaminergic and serotonergic neurons as well as octopaminergic, type II terminals at the neuromuscular junction. Surprisingly, DVMAT-A is co-expressed at type II terminals with the Drosophila vesicular glutamate transporter. Our data suggest that DVMAT-A functions as a vesicular transporter for DA, 5HT, and octopamine in vivo, and will provide a powerful invertebrate model for the study of transporter trafficking and regulation.     

The Effects of Silicone Fluid Additives and Silicone Elastomer Matrices on Barnacle Adhesion Strength

Barnacle adhesion strength was used to screen seventy-seven polydimethylsiloxane elastomeric coatings for fouling-release properties. The test coatings were designed to investigate the effect on barnacle adhesion strength of silicone fluid additive type, additive location, additive molecular weight, additive loading level, mixtures of additives, coating matrix type and coating fillers. The type of silicone fluid additive was the primary controlling factor in barnacle fouling-release. The type of silicone matrix in which the fluid resided was found to alter the effect on fouling-release. Two PDMS fluids, DMSC15 and DBE224, significantly reduced the adhesion strength of barnacles compared to unmodified elastomers. Optimum fouling-release performance was dependent on the interaction of fluid type and elastomeric matrix.     

K2P18.1 translates T cell receptor signals into thymic regulatory T cell development

It remains largely unclear how thymocytes translate relative differences in T cell receptor (TCR) signal strength into distinct developmental programs that driv...     

Bombesin-like peptides in human small cell carcinoma of the lung

The nature of bombesin-like immunoreactive peptides was studied in extracts of small cell carcinoma of the human lung. Three peaks, I, II and III, designated by their increasing retention times, were separated by reversed-phase high performance liquid chromatography (HPLC) with trifluoroacetic acid (TFA) as counter ion. None of the peaks corresponded to bombesin. Peak III was eluted at the same position as porcine gastrin releasing peptide (GRP) but was separated from it in another reversed-phase system using heptafluorobutyric acid (HFBA). Peak II material eluted in the position of bombesin in the HFBA system but not in the TFA system. The elution position of Peak I corresponded to that of the carboxyl terminal fragments of GRP, i.e. GRP18-27 and GRP19-27. This correspondence was observed in each of the reversed-phase and gel filtration systems used. The Peak III peptide was converted to peak I after incubation with trypsin. It was reasoned that this conversion could be one of the steps in the processing of bombesin-like peptides in human small cell carcinoma.     

Enhancement in the viability and biosensing activity of freeze-dried recombinant bioluminescent bacteria

The genetically-engineeredEscherichia coli strain, DPD2540, which contains afabA::luxCDABE fusion gene, gives a bioluminescent output when membrane fatty acid synthesis is needed. For more practical application of this strain in the field as biosensor, freeze-drying was adopted. A 12% sucrose solution with Luria-Bertani (LB) broth, as determined by the viability after freeze-drying, was found to be the most effective composition for lyophilization solution among various compositions tested. Rapid freezing with liquid nitrogen also gave the best viability after freeze-drying as compared to samples frozen at −70°C and −20°C. The biosensing activities of the cells showed a greater sensitivity when the cells from the exponential phase were freeze-dried. Finally, the optimum temperature for use of the freeze-dried cells in the biosensor field was determined.     

Plasma membrane block to sperm entry occurs in mouse eggs upon parthenogenetic activation

The ability of parthenogenetically activated mouse eggs to establish a plasma membrane (PM) block to sperm penetration was studied. Zona-free eggs preloaded with Hoechst 33342 were activated by exposure to ethanol or OAG (1-oleoyl-2-acetyl-sn-glycerol) and inseminated after different periods. Eggs challenged with sperm at 30- or 60-min postactivation displayed a fertilization frequency significantly lower than that of control eggs. Conversely, when insemination was carried out at 120-min postactivation, the proportion of fertilized eggs was equivalent to that observed in the control group. Moreover, we report that when the eggs were induced to resume meiosis without any notable loss of CGs (egg exposure to OAG at 100 μM external Ca²⁺ or to heat shock), a normal ability to be penetrated was recorded at 30-min postactivation. Similar behaviour was exhibited by eggs that underwent a CG exocytosis close to that triggered by sperm in absence of nuclear activation (microinjection of inositol 1,4,5-trisphosphate into the egg at 1 μM cytosolic concentration). Present data support the conclusion that parthenogenetically activated mouse eggs are capable of a transitory PM block response that requires both CG exocytosis and meiosis resumption to occur. © 1994 Wiley-Liss, Inc.     

Use of gas chromatography-mass spectrometry/solid phase microextraction for the identification of MVOCs from moldy building materials

Gas chromatography-mass spectrometry/solid phase microextraction (GC-MS/SPME) was applied to identify microbial volatile organic compounds (MVOCs) in water-damaged, mold-infested building materials (gypsum board papers (n=2), mineral wool, and masonite) and in cultivated molds (Aspergillus penicillioides, Stachybotrys chartarum, and Chaetomium globosum). Three SPME fibers (65-microm PDMS-DVB, 75-microm Carboxen-PDMS, and 70-microm Carbowax-stableflex) designed for automated injection were used of which the latter showed best performance. A number of previously reported MVOCs were detected both in the building materials and the cultivated molds. In addition, methyl benzoate was identified both in the S. chartarum and A. penicillioides cultures and in the building materials. SPME combined with GC-MS may be a useful method for the determination of MVOCs emitted from mold-infested building materials.     

CaGE: cardiac gene expression knowledgebase

CaGE is a Cardiac Gene Expression knowledgebase we have developed to facilitate the analysis of genes important to human cardiac function. CaGE integrates the functionality of the LocusLink database with data from several human cardiac expression libraries, phenotypic data from OMIM and data from large-scale microarray gene expression studies to create a knowledgebase of gene expression in human cardiac tissue. The knowledgebase is fully searchable via the web using several intuitive query interfaces. Results can be displayed in several concise easy to navigate formats. AVAILABILITY: CaGE is located at http://www.cage.wbmei.jhu.edu