Ratiometric fluorescent sensing of pyrophosphate with sp³-functionalized single-walled carbon nanotubes.

Inorganic pyrophosphate is a key molecule in many biological processes from DNA synthesis to cell metabolism. Here we introduce sp3-functionalized (6,5) single-walled carbon nanotubes (SWNTs) with red-shifted defect emission as near-infrared luminescent probes for the optical detection and quantification of inorganic pyrophosphate. The sensing scheme is based on the immobilization of Cu2+ ions on the SWNT surface promoted by coordination to covalently attached aryl alkyne groups and a triazole complex. The presence of Cu2+ ions on the SWNT surface causes fluorescence quenching via photoinduced electron transfer, which is reversed by copper-complexing analytes such as pyrophosphate. The differences in the fluorescence response of sp3-defect to pristine nanotube emission enables reproducible ratiometric measurements in a wide concentration window. Biocompatible, phospholipid-polyethylene glycol-coated SWNTs with such sp3 defects are employed for the detection of pyrophosphate in cell lysate and for monitoring the progress of DNA synthesis in a polymerase chain reaction. This robust ratiometric and near-infrared luminescent probe for pyrophosphate may serve as a starting point for the rational design of nanotube-based biosensors.

Stochastic Formation of Quantum Defects in Carbon Nanotubes.

Small perturbations in the structure of materials significantly affect their properties. One example is single wall carbon nanotubes (SWCNTs), which exhibit chirality-dependent near-infrared (NIR) fluorescence. They can be modified with quantum defects through the reaction with diazonium salts, and the number or distribution of these defects determines their photophysics. However, the presence of multiple chiralities in typical SWCNT samples complicates the identification of defect-related emission features. Here, we show that quantum defects do not affect aqueous two-phase extraction (ATPE) of different SWCNT chiralities into different phases, which suggests low numbers of defects. For bulk samples, the bandgap emission (E11) of monochiral (6,5)-SWCNTs decreases, and the defect-related emission feature (E11*) increases with diazonium salt concentration and represents a proxy for the defect number. The high purity of monochiral samples from ATPE allows us to image NIR fluorescence contributions (E11 = 986 nm and E11* = 1140 nm) on the single SWCNT level. Interestingly, we observe a stochastic (Poisson) distribution of quantum defects. SWCNTs have most likely one to three defects (for low to high (bulk) quantum defect densities). Additionally, we verify this number by following single reaction events that appear as discrete steps in the temporal fluorescence traces. We thereby count single reactions via NIR imaging and demonstrate that stochasticity plays a crucial role in the optical properties of SWCNTs. These results show that there can be a large discrepancy between ensemble and single particle experiments/properties of nanomaterials.

Guanine Quantum Defects in Carbon Nanotubes for Biosensing.

Fluorescent single-wall carbon nanotubes (SWCNTs) are used as nanoscale biosensors in diverse applications. Selectivity is built in by noncovalent functionalization with polymers such as DNA. Recently, covalent functionalization was demonstrated by conjugating guanine bases of adsorbed DNA to the SWCNT surface as guanine quantum defects (g-defects). Here, we create g-defects in (GT)10-coated SWCNTs (Gd-SWCNTs) and explore how this affects molecular sensing. We vary the defect densities, which shifts the E11 fluorescence emission by 55 nm to a λmax of 1049 nm. Furthermore, the Stokes shift between absorption and emission maximum linearly increases with defect density by up to 27 nm. Gd-SWCNTs represent sensitive sensors and increase their fluorescence by >70% in response to the important neurotransmitter dopamine and decrease it by 93% in response to riboflavin. Additionally, the extent of cellular uptake of Gd-SWCNTs decreases. These results show how physiochemical properties change with g-defects and that Gd-SWCNTs constitute a versatile optical biosensor platform.

Decomposition of 2-chloroethylethylsulfide on copper oxides to detoxify polymer-based spherical activated carbons from chemical warfare agents.

For the decomposition of chemical warfare agents, a hybrid material concept was applied. This consists of a copper oxide-containing phase as a component with reactive functionality supported on polymer-based spherical activated carbon (PBSAC) as a component with adsorptive functionality. A corresponding hybrid material was prepared by impregnation of PBSAC with copper(II)nitrate and subsequent calcination at 673K. The copper phase exists predominantly as copper(I)oxide which is homogeneously distributed over the PBSAC particles. The hybrid material containing 16 wt.% copper on PBSAC is capable of self-detoxifying the mustard gas surrogate 2-chloroethylethylsulfide (CEES) at room temperature. The decomposition is related to the breakthrough behavior of the reactant CEES, which displaces the reaction product ethylvinylsulfide (EVS). This leads to a combined breakthrough of CEES and EVS. The decomposition of CEES is shown to occur catalytically over the copper-containing PBSAC material. Thus, the hybrid material can even be considered to be self-cleaning.

An apical-type trafficking pathway is present in cultured oligodendrocytes but the sphingolipid-enriched myelin membrane is the target of a basolateral-type pathway.

Myelin sheets originate from distinct areas at the oligodendrocyte (OLG) plasma membrane and, as opposed to the latter, myelin membranes are relatively enriched in glycosphingolipids and cholesterol. The OLG plasma membrane can therefore be considered to consist of different membrane domains, as in polarized cells; the myelin sheet is reminiscent of an apical membrane domain and the OLG plasma membrane resembles the basolateral membrane. To reveal the potentially polarized membrane nature of OLG, the trafficking and sorting of two typical markers for apical and basolateral membranes, the viral proteins influenza virus-hemagglutinin (HA) and vesicular stomatitis virus-G protein (VSVG), respectively, were examined. We demonstrate that in OLG, HA and VSVG are differently sorted, which presumably occurs upon their trafficking through the Golgi. HA can be recovered in a Triton X-100-insoluble fraction, indicating an apical raft type of trafficking, whereas VSVG was only present in a Triton X-100-soluble fraction, consistent with its basolateral sorting. Hence, both an apical and a basolateral sorting mechanism appear to operate in OLG. Surprisingly, however, VSVG was found within the myelin sheets surrounding the cells, whereas HA was excluded from this domain. Therefore, despite its raft-like transport, HA does not reach a membrane that shows features typical of an apical membrane. This finding indicates either the uniqueness of the myelin membrane or the requirement of additional regulatory factors, absent in OLG, for apical delivery. These remarkable results emphasize that polarity and regulation of membrane transport in cultured OLG display features that are quite different from those in polarized cells.

Differential and cell development-dependent localization of myelin mRNAs in oligodendrocytes.

In oligodendrocytes (OLG), the mRNAs for the various myelin proteins localize to different intracellular sites. Whereas the confinement of myelin basic protein (MBP) mRNA to the processes of the cell has been well established, we demonstrate that most other myelin mRNA species are mainly present in the perinuclear region. Using in situ hybridization of cultured rat OLG we found that mRNAs are localized to at least three different locations: 1) to the perinuclear region [myelin-associated glycoprotein (MAG) mRNA]; 2) mainly to the processes (the mRNA for the 14-kDa isoform of MBP); and 3) to both the perinuclear region and the primary processes [2',3'-cyclic nucleotide phosphodiesterase (CNPase) and proteolipid protein (PLP) mRNAs]. Thus, depending on their primary structure, the mRNA species in OLG either remain near the nucleus or localize to primary or secondary processes before their translation. The myelin mRNA localization correlates well with that of the proteins encoded in them, as demonstrated by immunocytochemistry. Since different isoforms of MBP have different locations in transfected HeLa cells (Staugaitis et al.: J Cell Biol 110:1719-1727, 1990), we also have investigated the localization of the various mRNAs in OLG, using exon 2-minus and exon 2-specific probes in situ hybridization. The exon 2-minus MBP mRNAs are transported far into the processes, whereas exon 2-specific mRNA was only detected in the cell body. This suggests that sorting and trafficking of MBP mRNA are regulated by the presence or absence of the exon 2 sequence. Furthermore, during maturation of OLG, exon 2-plus mRNAs disappear, whereas exon 2-minus mRNAs increase. The developmentally regulated expression of exon 2-plus transcripts suggest a role of their protein products in differentiation rather than in myelination.

Outstations of the Golgi complex are present in the processes of cultured rat oligodendrocytes.

Primary cultures of rat oligodendrocytes were incubated with a fluorescent sphingolipid precursor, 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoylceramide+ ++ (C6-NBD-ceramide). This compound is known to stain the Golgi complex specifically. Within 30 min of incubation at 37 degrees C most of the C6-NBD-ceramide was incorporated into the perinuclear Golgi system, as revealed by conventional and confocal laser fluorescence microscopy. Interestingly, C6-NBD-ceramide was found to accumulate also in smaller, oval-shaped structures in many of the processes, at distances up to 30 microns from the nucleus. This implies the possibility that these structures are Golgi (-derived) complexes. Indeed, after incubation of oligodendrocytes with C6-NBD-ceramide and rhodamine-labeled transferrin both fluorescent labels colocalized in the Golgi system of the cell body as well as in the structures in the processes. Additional support for the Golgi character of these structures was obtained by transmission electron microscopy. Particularly in oligodendrocytes cocultured with neurons, many Golgi structures were present all over the processes. The results lead us to conclude that, in the oligodendrocyte, the Golgi complex does not only reside in the perikaryon, but also in the processes. One can speculate that a polarized biosynthetic activity, involving the presence of the Golgi near the site of myelin synthesis, may be advantageous to the oligodendrocyte for assembly and/or repair of the myelin membrane at the distal end of the processes.

Assignment of the gene coding for human cytochrome c oxidase subunit VIb to chromosome 19, band q13.1, by fluorescence in situ hybridisation.

A cloned, 40 kb, genomic DNA fragment, containing the last exon of the gene for human cytochrome c oxidase subunit VIb and its flanking sequences, was used as a probe to localize the subunit VIb gene on human metaphase chromosomes. The probe was labelled with Bio-11-dUTP and detected by fluorescence. Subsequent R-banding indicated that the cytochrome c oxidase subunit VIb gene is localized in band 19q13.1, extending the evidence that the human nuclear genes of cytochrome c oxidase are not clustered.

Identification of three human pseudogenes for subunit VIb of cytochrome c oxidase: a molecular record of gene evolution.

Three pseudogenes for the nuclear-encoded subunit VIb of cytochrome c oxidase (COX) were isolated by screening a human genomic library with cloned human cDNA coding for COX subunit VIb. The nucleotide sequences of the pseudogenes, designated psi COX6b-1, psi COX6b-2 and psi COX6b-3, were determined. Pseudogene psi COX6b-1 bears all the hallmarks of a processed pseudogene and diverged from the parental gene after the divergence of man and cow. Alu repetitive elements were integrated into the structural sequences of the other two pseudogenes. Comparison with the human and bovine cDNA sequences encoding COX subunit VIb suggests that psi COX6b-2 and psi COX6b-3 were formed earlier in evolution than psi COX6b-1. Genomic Southern analysis indicated that a few more pseudogenes for COX subunit VIb are likely to be present in the human genome. Identical nt differences with respect to the human cDNA sequence in the pseudogenes provide some clues on the evolution of the ancestral gene coding for COX subunit VIb.

Nucleotide sequence of the last exon of the gene for human cytochrome c oxidase subunit VIb and its flanking regions.

A human genomic clone encompassing the last exon of the gene for cytochrome c oxidase subunit VIb and a human genomic clone containing the most distal end of this gene were characterized. The last exon of the gene codes for the 17 C-terminal amino acid residues of the subunit and the 3' noncoding region. Downstream from the gene we found a single base difference between the DNA sequences of the two genomic clones. An inverted Alu dimer repeat was identified further downstream.

Isolation of cDNAs encoding subunit VIb of cytochrome c oxidase and steady-state levels of coxVIb mRNA in different tissues.

A full-length cDNA clone specifying the nuclear-encoded subunit VIb of human cytochrome c oxidase (COX) was isolated from a human skeletal muscle cDNA expression library. This was done with antiserum directed against the group of subunits VIa, b and c of bovine heart COX. A potential ribosome-binding site was located immediately upstream from the initiation codon. The predicted amino acid sequence revealed 85% similarity with the corresponding subunit of bovine heart COX. Subunit VIb lacks a cleavable presequence for mitochondrial addressing. We assume that there are no tissue-specific isoforms of subunit VIb, since (i) in a Northern blot experiment a single hybridizing band of approx. 500 nucleotides was demonstrated in RNA from liver, skeletal muscle, MOLT-4 cells and fibroblasts and (ii) a full-length cDNA clone with an identical sequence was isolated from a human liver cDNA library. Steady-state levels of the coxVIb transcript were different in the tissues examined.

Evidence that the neuroleptic fluphenazine replaces Ca(2+) and adjusts the noradrenaline induced cyclic adenosine monophosphate synthesis in the rat retina.

UNLABELLED: Intact rat retinae were incubated in Krebs-Ringer media with noradrenaline (NA) in the presence (0.75 mM) or absence of extracellular Ca(2+) and at relatively high (10 mM) or low (1 mM) theophylline concentrations. Depending on the incubation conditions we found that the neuroleptic fluphenazine (FLU) affected cAMP-synthesis separately from cAMP-degradation of the NA-cAMP system in the retina. The main results were: At a relatively high theophylline concentration of 10 mM, where cAMP synthesis alone is operative, and at 0.75 Ca(2+) we measured with 50 ?M NA a NA-response of 110 pmol cAMP/mg prot. At a low theophylline concentration of 1 mM and again at 0.75 mM Ca(2+) both cAMP-synthesis and -breakdown are operative. In this condition we found the NA-response of 26 pmol cAMP/mg prot. to be raised by 10 ?M FLU to 130 pmol cAMP/mg prot. This enhancing effect might be due to inhibition of degradation of NA-induced cAMP by FLU. In the absence of extracellular calcium and again at 10 mM theophylline, 10 ?M FLU raised the NA response nearly 4-fold from 42 pmol cAMP/mg prot. to 153 pmol cAMP/mg prot. The lowest effective concentration for obtaining this enhancing effect was 10 ?M FLU and the effect is characterized by an apparent K(m) of 0.5 ?M. The use of 10 mM theophylline in this condition suggests that this FLU-Ca(2+) effect is confined to the synthesis part of the NA-cAMP system. The effect points to a replacement of an intramembraneous Ca(2+) function by FLU. IN CONCLUSION: our results suggest that FLU inhibits degradation of NA-induced synthesis of cAMP and that the neuroleptic renders the NA-response less dependent on extracellular Ca(2+).

Extracellular potassium controls responsiveness of the noradrenergic cAMP system in the rat retina to fluphenazine.

The effects of fluphenazine (FLU) on the noradrenaline (NA) induced cAMP-synthesis in intact rat retinae were studied as a function of extracellular K(+)- and Ca(2+)-ions. Thus NA-induced cAMP levels were measured after incubating intact rat retinae with 50 ?M NA in the presence or absence of FLU and in the presence of 1 or 10 mM theophylline. Results were: (1) Experimental condition a: standard NA-responses were measured after incubating retinae at 0.75 mM Ca(2+), at 10 mM theophylline, at 10 ?M FLU and at 2 and 0 mM K(+). FLU does not affect the NA-response at 2 mM K(+) significantly; however, it inhibits the NA-response at 0 mM K(+) in this condition. (2) Experimental condition b: NA-responses were measured after incubating retinae at 0.125 mM Ca(2+), 10 mM theophylline, 10 ?M FLU and at 2 and 0 mM K(+). At 2 mM K(+) FLU replaces a Ca(2+) function probably connected with the synthesis part of the NA-cAMP system and NA-responses in this low Ca(2+) condition are consequently enhanced by FLU; however, FLU inhibits the NA-response at 0 mM K(+) in this condition. (3) Experimental condition c: NA-responses were measured after incubating retinae at 0.75 mM Ca(2+), 1 mM theophylline, 10 ?M FLU and at 2 and 0 mM K(+). At 2 mM K(+) FLU enhances the NA-response by further inhibition of the degradation part of the NA-cAMP system; FLU inhibits the NA-response at 0 mM K(+) in this condition. (4) The inhibitions of the NA-responses by FLU at 0 mM K(+) in all three conditions a, b and c showed an apparent K(m) of 1 ?M. (5) Low concentrations of K(+) (0.4-0.8 mM) maintain the property of FLU to enhance the NA-responses at condition b (0.125 mM Ca(2+)) and at condition c (1 mM theophylline). Results suggest that the activation of NA-receptor coupled adenylate cyclases (NA-AC-ases) by NA, resulting in activation of phosphodiesterase activity by the NA-elevated cAMP-levels, is sustained by (a) membraneous factor(s) connected to the NA-receptor. This (these) factor(s) is (are) switched off in the absence of K(+). Evidence has been presented, that Ca(2+) and FLU do not have access to this intramembraneous factor-enzyme activating moiety of the NA-cAMP system at 0 mM K(+). Between 0.4 and 0.8 mM K(+) the factor-enzyme-NA-receptor complex is still intact.