Detection of bioterror agents in air samples using real-time PCR.

AIMS: To use real-time PCR for the detection of bacterial bioterror agents in a liquid air sample containing potential airborne interferences, including bacteria, without the need for DNA extraction. METHODS AND RESULTS: Bacteria in air were isolated after passive sedimentation onto R2A agar plates and characterized by 16S rRNA sequencing. Real-time PCR was used to identify different bacterial bioterror agents in an artificial air sample consisting of a liquid air sample and a mixture of miscellaneous airborne bacteria showing different colony morphology on R2A agar plates. No time-consuming DNA extraction was performed. Specifically designed fluorescent hybridization probes were used for identification. CONCLUSIONS: Fourteen different bacterial genera were classified by 16S rRNA gene sequencing of selected bacterial colonies showing growth on R2A agar plates. Real-time PCR amplification of all the bacterial bioterror agents was successfully obtained in the artificial air sample containing commonly found airborne bacteria and other potential airborne PCR interferences. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacterial bioterror agents can be detected within 1 h in a liquid air sample containing a variety of commonly found airborne bacteria using real-time PCR. Airborne viable bacteria at Kjeller (Norway) were classified to the genera level using 16S rRNA gene sequencing.

Phosphorylation of dynamin I and synaptic-vesicle recycling.

In nerve terminals, neurotransmitters are packaged in synaptic vesicles, and released by exocytosis. Empty synaptic vesicles are rapidly recycled for reuse by endocytosis. Much progress has been made in identifying the proteins involved in synaptic-vesicle trafficking, but the mechanism and regulation of endocytosis have largely remained an enigma. One approach to defining regulatory proteins that might be involved is to study stimulus-dependent phosphorylation events in nerve terminals. This has led to the identification of dephosphin, which is quantitatively dephosphorylated by nerve-terminal depolarization. Sequencing reveals that dephosphin is identical with dynamin I, a GTP-binding protein that functions in endocytosis. Phosphorylation and dephosphorylation of nerve-terminal dynamin I/dephosphin regulates its intrinsic GTPase activity in parallel with the regulation of synaptic-vesicle recycling. Therefore, phosphorylation and dephosphorylation of dynamin I might provide a Ca(2+)-dependent switch for endocytosis in the synaptic-vesicle pathway.

Uptake of L-glutamate into synaptic vesicles: competitive inhibition by dyes with biphenyl and amino- and sulphonic acid-substituted naphthyl groups.

The specificity of the vesicular L-glutamate carrier was characterized using dyes with biphenyl and amino- and sulphonic acid substituted naphthyl groups, structurally similar to the specific vesicular L-glutamate inhibitor Evans Blue. The dye Trypan Blue was the most potent inhibitor; the IC50 value was determined to be 49 nM. Naphthol Blue Black, Reactive Blue 2, Benzopurpurin 4B, Ponceau SS, Direct Blue 71 and Acid red 114 were also highly potent inhibitors with IC50 values from 330 to 1670 nM (series 1). The dyes were competitive inhibitors of vesicular glutamate uptake, and acted therefore on the glutamate transporter. Their IC50 values for the vesicular uptake of gamma-aminobutyric acid (GABA) were all higher than 20 microM. They had no effect on synaptosomal uptake of glutamate. Furthermore, we have also found several other dyes with IC50 values for the vesicular uptake of glutamate ranging between 1 and 30 microM and for gamma-aminobutyric acid higher than 50 microM (series 2). The most potent inhibitor Trypan Blue contains a biphenyl group, linked by azo groups to side chains containing sulphonic, amino and/or hydroxyl groups coupled to a naphthalene ring system. Trypan Blue and Evans Blue are by molecular mechanics, shown to have planar structures with conjugated double bonds throughout the structure. The other dyes, which were less effective, had phenyl and/or naphthalene groups linked by an azo group. We have also tested a series of amino and/or hydroxyl naphthalene di-/sulphonic acids that correspond to the side chains of the most potent dyes, but they had no effect on glutamate nor on gamma-aminobutyric acid uptake. We conclude that the inhibitory action of these compounds is strictly dependent of the complete molecule.

Inhibition of gamma-aminobutyrate and glycine uptake into synaptic vesicles.

The substrate specificity of vesicular GABA and glycine uptake was studied in vesicle fractions from brain and spinal cord, respectively. Glycine, beta-alanine and gamma-vinyl-GABA were competitive inhibitors of the GABA uptake were competitive inhibitors of the GABA uptake by synaptic vesicles in brain. Likewise GABA and beta-alanine turned out to be competitive inhibitors of vesicular uptake of glycine in spinal cord. The apparent K1 values were in the same range as the respective Km values for the transport systems. Accumulation of different amino acids were examined, and the structurally related amino acids GABA, beta-alanine and glycine were all taken up by both vesicle fractions. In the present study, we suggest that there are similarities in the vesicular transporters for GABA and glycine, and the two amino acids are probably taken up into the same vesicle population. The key factor in differentiating between GABA and glycine as transmitters in the terminals could be the synthesis and the high-affinity synaptosomal uptake.

The effect of arachidonic acid and free fatty acids on vesicular uptake of glutamate and gamma-aminobutyric acid.

The manner in which arachidonic acid and other free fatty acids influence the vesicular uptake of glutamate and gamma-aminobutyric acid (GABA) has been investigated. The cis-polyunsaturated fatty acid arachidonic acid (20:4), eicosapentanoic acid (20:5) and linolenic acid (18:3) at 150 nmol/mg protein (50 microM) inhibited the vesicular uptake of glutamate and GABA more than 70%. Reduced inhibition of vesicular uptake was seen with the cis-monounsaturated fatty acid oleic acid (18:1) and the trans-mono-unsaturated fatty acid elaidic acid (18:1). The saturated fatty acids stearic acid (16:0) and arachidic acid (20:0) had no significant effect on the uptake. The inhibition of vesicular uptake by arachidonic acid was prevented by the addition of fatty acid free bovine serum albumin. Arachidonic acid inhibited in a dose-dependent manner the generation of the transmembrane pH gradient of the synaptic vesicles. This inhibition was proportional to the inhibition of the vesicular uptake of glutamate and GABA. The saturated fatty acid arachidic acid showed no inhibition of delta pH generation. Arachidonic acid at 200 nmol/mg of protein did not increase the uptake-independent leakage of glutamate and GABA from the vesicles, showing that the effect of arachidonic acid is not caused by an unspecific detergent effect. These results suggest that arachidonic acid and other polyunsaturated fatty acids are acting like proton-ionophores on the vesicular uptake of these neurotransmitters. This finding may have implications for the increased fatty acid concentration during pathological conditions like ischemia and in long term potentiation.

Regional distribution of gamma-aminobutyrate and L-glutamate uptake into synaptic vesicles isolated from rat brain.

The ATP-dependent uptake of GABA and L-glutamate into synaptic vesicles isolated from 4 different regions of the rat brain was studied. The regional distribution of the vesicular uptake was related to the Na+-dependent synaptosomal uptake, which, as a first approximation, corresponds to the distribution of GABAergic and glutamatergic terminals. The ratio found between the vesicular GABA and L-glutamate uptake varied between 1.3 and 6.2. This indicates that GABA and L-glutamate are taken up into different vesicle populations.

Inhibition of L-glutamate uptake into synaptic vesicles.

The effects of different agents similar in structure to glutamate were tested for inhibition of the vesicular uptake of L-glutamate. Kainate and L-homocysteate turned out to be non-competitive inhibitors of the L-glutamate uptake. Kainate was not taken up by the vesicle fraction. The vesicular uptake of gamma-aminobutyric acid (GABA) was also inhibited by kainate and L-homocysteate. Kynurenate, on the other hand, strongly inhibited the uptake of L-glutamate, whereas the uptake of GABA was hardly affected. L-alpha-Aminoadipate and D-glutamate inhibited the uptake of L-glutamate, whereas L- and D-aspartate and L-cysteate only weakly inhibited the uptake of L-glutamate. GABA, glycine, L-serine and taurine did not inhibit the uptake of L-glutamate.

Depolarization of cerebellar granule cells increases phosphorylation of rabphilin-3A.

Studies performed over the past several years have provided evidence that phosphorylation of proteins is important in the regulation of neurotransmitter release. In this study, it is shown that rabphilin-3A is present in cerebellar granule cells as a phosphoprotein, by using 32P-labeling of cerebellar granule cells, immunoprecipitation, phosphoamino acid analysis, and phosphopeptide mapping. The level of phosphorylation was increased (224 +/- 13%) (mean +/- SEM) on depolarization of the cells with K+ (56 mM) in the presence of external Ca2+ (1 mM). Stimulation of protein kinase C with a phorbol ester (phorbol 12,13-dibutyrate) also enhanced the phosphorylation of rabphilin-3A (217 +/- 21%). Inhibitors of Ca2+/calmodulin-stimulated protein kinases or protein kinase C reduced the depolarization-enhanced phosphorylation of rabphilin-3A, indicating that rabphilin-3A is one of the targets for Ca2+-activated protein kinases in the nerve terminal. Costimulation of cells with phorbol 12,13-dibutyrate and K+ depolarization produced an increased level of phosphorylation of rabphilin-3A compared with either stimulus alone (287 +/- 61%). Phosphoamino acid analysis showed that serine was the main phosphorylated residue. A slight increase in the threonine phosphorylation could also be detected, whereas tyrosine phosphorylation could not be detected at all. These results suggest that rabphilin-3A is phosphorylated in vivo and undergoes synaptic activity-dependent phosphorylation during Ca2+-activated K+ depolarization.

Uptake of gamma-aminobutyric acid by a synaptic vesicle fraction isolated from rat brain.

Gamma-Aminobutyric acid (GABA) was taken up by a MgATP-dependent mechanism into synaptic vesicles isolated by hypoosmotic shock and density gradient centrifugation. The properties of the vesicular uptake differed clearly from those of synaptosomal and glial uptake, both with respect to Na+, Mg2+, and ATP dependence and with respect to response to general GABA uptake inhibitors such as nipecotic acid, diaminobutyric acid, and beta-alanine. The uptake showed a Km of 5.6 mM and a net uptake rate of 1,500 pmol/min/mg of protein. It is suggested that the vesicular uptake of GABA is driven by an electrochemical proton gradient generated by a Mg2+-ATPase.

Amino acid neurotransmission: dynamics of vesicular uptake.

Glutamate, GABA and glycine, the major neurotransmitters in CNS, are taken up and stored in synaptic vesicles by a Mg(2+)-ATP dependent process. The main driving force for vesicular glutamate uptake is the membrane potential, whereas both the membrane potential and the proton gradient contribute to the uptake of GABA and glycine. Glutamate is taken up by a specific transporter with no affinity for aspartate. Evans blue and related dyes are competitive inhibitors of the uptake of glutamate. GABA, beta-alanine, and glycine are taken up by the same family of transporter molecules. Aspartate, taurine, and proline are not taken up by any synaptic vesicle preparations. It is suggested that vesicular uptake and release are characteristics that identify these amino acids as neurotransmitters. We also discuss that "quanta" in the brain are not necessarily related the content of neurotransmitter in the synaptic vesicles, but rather to postsynaptic events.

Transport of gamma-aminobutyrate and L-glutamate into synaptic vesicles. Effect of different inhibitors on the vesicular uptake of neurotransmitters and on the Mg2(+)-ATPase.

The uptakes of gamma-aminobutyrate (GABA) and L-glutamate into synaptic vesicles isolated from rat brain were compared with respect to the effects of 4-acetamido-4'-isothiocyanostilbene-2,2'- disulphonic acid (SITS), 4,4'-di-isothiocyanostilbene-2,2'-disulphonic acid (DIDS) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (N144), agents known to block anion channels. The uptake of glutamate was inhibited by low micromolar concentrations of SITS, DIDS and N144. GABA uptake was much less sensitive to these agents than was glutamate uptake. SITS and N144 inhibited the vacuolar H(+)-ATPase of synaptic vesicles to a smaller extent than the glutamate uptake. The uptake of GABA was not affected by the permeant anions Cl- and Br-, whereas the uptake of glutamate was highly stimulated by low concentrations of these ions. The uptakes of both glutamate and GABA were inhibited by similar, but not identical, concentrations of the lipophilic anion SCN-.

Phosphorylation of rabphilin-3A by Ca2+/calmodulin- and cAMP-dependent protein kinases in vitro.

Regulation of neurotransmitter release is thought to involve modulation of the release probability by protein phosphorylation. In order to identify novel targets for such regulatory processes, we have studied the phosphorylation of rabphilin-3A in vitro. Rabphilin-3A is a synaptic vesicle protein that interacts with rab3A in a GTP-dependent manner and binds Ca2+ in a phospholipid-dependent manner. Here we show that rabphilin-3A is an efficient substrate for Ca2+/calmodulin-dependent protein kinase II, which phosphorylates rat rabphilin-3A at residue 234 and 274, and for cAMP-dependent protein kinase, which phosphorylates rat rabphilin-3A at residue 234. This identifies the middle region of rabphilin-3A situated between the N-terminal rab3A-binding sequences and the C-terminal C2-domains involved in Ca2+/phospholipid binding as a regulatory domain. Thus, rabphilin-3A is a second phosphoprotein on synaptic vesicles that, similar to synapsin I, may integrate phosphorylation signals from multiple protein kinase signaling pathways in the cell.

Uptake of L-glutamate into rat brain synaptic vesicles: effect of inhibitors that bind specifically to the glutamate transporter.

In this study we have described a series of new and potent inhibitors of the vesicular uptake of glutamate. The two most efficient inhibitors were the dyes Evans blue and Chicago Skye Blue 6B, which are structurally related to glutamate and were competitive inhibitors in the nanomolar range. The anion channel blocker 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (SITS) and the diuretics furosemide and bumetanide are inhibitors of chloride transport in other organs but were competitive inhibitors of glutamate and noncompetitive with respect to chloride ions. Evans blue, Chicago Skye Blue 6B, SITS, furosemide, and bumetanide are all large organic acids with two centers of negative charge and an electron-donating group at close vicinity of the negative charge at physiological pH. The inhibition of the glutamate uptake with these inhibitors was noncompetitive with respect to Cl-. The inhibitors, therefore, probably interact directly with the glutamate carrier. Bafilomycin A1, which is a specific vacuolar ATPase inhibitor, was used as a control and inhibited the vesicular dopamine, glutamate, and GABA uptake to the same extent. None of the inhibitors had any effect on the plasma membrane carrier, which is therefore clearly different from the vesicular carrier.

Comparison of the properties of gamma-aminobutyric acid and L-glutamate uptake into synaptic vesicles isolated from rat brain.

Rat brain synaptic vesicles exhibit ATP-dependent uptake of gamma-[3H]amino-n-butyric acid ([3H]GABA) and L-[3H]glutamate. After hypotonic shock, the highest specific activities of uptake of both L-glutamate and GABA were recovered in the 0.4 M fraction of a sucrose gradient. The uptakes of L-glutamate and GABA were inhibited by similar, but not identical, concentrations of the mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrazone and the ionophores nigericin and gramicidin, but they were not inhibited by the K+ carrier valinomycin. N,N'-Dicyclohexyl-carbodiimide and N-ethylmaleimide, Mg2+-ATPase inhibitors, inhibited the GABA and L-glutamate uptakes similarly. Low concentrations of Cl- stimulated the vesicular uptake of L-glutamate but not that of GABA. The uptakes of both L-glutamate and GABA were inhibited by high concentrations of Cl-. These results indicate that the vesicular GABA and L-glutamate uptakes are driven by an electrochemical proton gradient generated by a similar Mg2+-ATPase. The vesicular uptake mechanisms are discussed in relation to other vesicle uptake systems.

Uptake of glycine into synaptic vesicles isolated from rat spinal cord.

Glycine was taken up by a synaptic vesicle fraction from spinal cord in a Mg-ATP-dependent manner. The accumulation of glycine was inhibited by carbonyl cyanide-m-chlorophenylhydrazone (CCCP) and nigericin, agents known to destroy the proton gradient across the vesicle membrane. Vesicular uptake of glycine was clearly different from synaptosomal uptake, with respect to both the affinity constant and the effect of Na+, ATP, CCCP, and temperature. Oligomycin and strychnine did not inhibit the vesicular uptake, showing that neither mitochondrial H(+)-ATPase nor binding to strychnine-sensitive glycine receptors was involved. It is suggested that the vesicular uptake of glycine is driven by a proton gradient generated by a Mg2(+)-ATPase. A low concentration of Cl- had little effect on the uptake of glycine, whereas the uptake of glutamate in the same experiment was highly stimulated. High concentrations of gamma-amino-n-butyric acid and beta-alanine inhibited vesicular glycine uptake, but glutamate did not. Accumulation of glycine was found to be fourfold higher in a spinal cord synaptic vesicle fraction than in a vesicle fraction from cerebral cortex.

The primary structure of the variable region of an immunoglobin IV light-chain amyloid-fibril protein (AL GIL).

The primary structure of the variable region of an amyloid-fibril protein GIL of immunoglobulin lambda-light-chain origin (AL) was determined. The AL protein obtained from the fibrils in the spleen of a 54-year-old man with primary systemic amyloidosis could be assigned to subgroup IV of the lambda variable-region sequence. About 50% of the protein was found to be truncated in the N-terminus and lacked the first six amino acid residues. The polypeptides consisted of about 146 amino acid residues and contained traces of carbohydrate. An acceptor site for N-glycosylation was found in positions 90-93, but no glycopeptide could be isolated. Comparison of the amino acid sequence of AL protein GIL with that of the only Bence-Jones protein of subgroup IV previously studied revealed a sequence homology of 89%. A similar comparison made with other AL proteins gave sequence homologies below 66%.

Relative properties and localizations of synaptic vesicle protein isoforms: the case of the synaptophysins.

Synaptophysins are abundant synaptic vesicle proteins present in two forms: synaptophysin, also referred to as synaptophysin I (abbreviated syp I), and synaptoporin, also referred to as synaptophysin II (abbreviated syp II). In the present study, the properties and localizations of syp I and syp II were investigated to shed light on their relative functions. Our results reveal that syp II, similar to syp I, is an abundant, N-glycosylated membrane protein that is part of a heteromultimeric complex in synaptic vesicle membranes. Cross-linking studies indicate that syp II is linked to a low-molecular-weight protein in this complex as has been observed before for syp I. Furthermore, after transfection into CHO cells, syp II, similar to syp I, is targeted to the receptor-mediated endocytosis pathway. Immunocytochemistry of rat brain sections reveals that syp II expression is highly heterogeneous, with high concentrations of syp II only in selected neuronal populations, whereas syp I is more homogeneously expressed in most nerve terminals. In general, nerve terminals expressing syp II also express syp I. In addition to high levels of syp II observed in selected neurons, a rostrocaudal gradient of syp II expression was observed in the cerebellar cortex. Immunoelectron microscopy confirmed that syp II is localized to synaptic vesicles. Immunoprecipitations of synaptic vesicles from rat brain with antibodies to syp I demonstrated that syp II is colocalized with syp I on the same vesicles. However, after detergent solubilization, no coimmunoprecipitations of the two proteins were observed, suggesting that they are not complexed with each other although they are on the same vesicles. Together our results demonstrate that syp I and syp II have similar properties and are present on the same synaptic vesicles but do not coassemble. The presence of the two proteins in the same nerve terminal suggests that they have similar but nonidentical functions and that the relative abundance of the two proteins may contribute to the functional heterogeneity of nerve terminals.

Phosphorylation of synaptotagmin I by casein kinase II.

Synaptotagmin I is an abundant synaptic vesicle protein that binds Ca2+ in a phospholipid-dependent manner and is thought to function in synaptic vesicle exocytosis. We have now studied the phosphorylation of synaptotagmin I. Synaptotagmin I is one of the major substrates in brain for casein kinase II, which phosphorylates synaptotagmin at a single threonine. The phosphorylation site was mapped using recombinant proteins to threonine 128 of synaptotagmin I, which is located in the sequence between the transmembrane region and the C2 domain repeats of synaptotagmin I. The phosphorylation site of synaptotagmin I is also present in synaptotagmin II and is evolutionarily conserved between different species. Preceding the phosphorylation site, synaptotagmins I and II contain a lysine-rich sequence. Casein kinase II phosphorylation of many substrates is strongly stimulated by the addition of polylysine, but phosphorylation of synaptotagmin I by casein kinase II is not. In recombinant proteins, removal of the lysine-rich sequence of synaptotagmin I makes its phosphorylation dependent on exogenous polylysine, suggesting that the lysine-rich sequence in synaptotagmin serves as an endogenous polylysine stimulation signal for casein kinase II. Our data demonstrate that synaptotagmin I is an efficient substrate for casein kinase II at a conserved site with a possible modulatory role in nerve terminal function.

Differential expression and regulation of multiple dynamins.

Dynamin is a GTP-, microtubule-, and phospholipid-binding protein that is expressed primarily in brain. In Drosophila, the shibire gene encodes a homologue of dynamin; mutations in this gene result in a defect in endocytosis, suggesting a function for dynamin in endocytic membrane traffic. In the present study we show that there are at least two distinct dynamin genes in mammals whose products are referred to as dynamins I and II. The two dynamins are similar to each other (79% identity) and are both equally homologous to the Drosophila shibire gene product (66% identity). The highest degree of identity between dynamins is observed in their N-terminal halves, whereas their C termini exhibit little homology. Transcripts of both dynamin genes are subject to at least two alternative splicing events, the first of which is identically found in both dynamins, whereas the second site of alternative splicing is different between the two types of dynamins. The first alternatively spliced sequence of the dynamins consists of an interior region that is present in two distinct but homologous forms in both dynamins, suggesting alternative use of exons in both genes at identical positions. The second site of alternative splicing results in the generation of different C termini in dynamin I and in the inclusion or exclusion of an interior four-amino acid sequence in dynamin II. The two dynamins exhibit remarkable differences in their tissue distribution and regulation. Dynamin I is almost exclusively expressed in the central nervous system. Conversely, dynamin II is expressed ubiquitously in all tissues tested. Previous studies revealed that the GTPase activity of dynamin I is regulated by phosphorylation by protein kinase C in nerve terminals. Expression of dynamins I and II by transfection in COS cells demonstrates that only dynamin I but not dynamin II is a substrate for protein kinase C. Our data suggest a specialization in the endocytic functions and the regulation of dynamins between neural and non-neural tissues in mammals.