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1.
Nat Commun ; 8(1): 188, 2017 08 04.
Article in English | MEDLINE | ID: mdl-28775280

ABSTRACT

Sensors using nitrogen-vacancy centers in diamond are a promising tool for small-volume nuclear magnetic resonance (NMR) spectroscopy, but the limited sensitivity remains a challenge. Here we show nearly two orders of magnitude improvement in concentration sensitivity over previous nitrogen-vacancy and picoliter NMR studies. We demonstrate NMR spectroscopy of picoliter-volume solutions using a nanostructured diamond chip with dense, high-aspect-ratio nanogratings, enhancing the surface area by 15 times. The nanograting sidewalls are doped with nitrogen-vacancies located a few nanometers from the diamond surface to detect the NMR spectrum of roughly 1 pl of fluid lying within adjacent nanograting grooves. We perform 1H and 19F nuclear magnetic resonance spectroscopy at room temperature in magnetic fields below 50 mT. Using a solution of CsF in glycerol, we determine that 4 ± 2 × 1012 19F spins in a 1 pl volume can be detected with a signal-to-noise ratio of 3 in 1 s of integration.Nitrogen vacancy (NV) centres in diamond can be used for NMR spectroscopy, but increased sensitivity is needed to avoid long measurement times. Kehayias et al. present a nanostructured diamond grating with a high density of NV centres, enabling NMR spectroscopy of picoliter-volume solutions.


Subject(s)
Diamond , Magnetic Resonance Spectroscopy , Nanostructures
2.
Rev Sci Instrum ; 82(2): 023118, 2011 Feb.
Article in English | MEDLINE | ID: mdl-21361584

ABSTRACT

We report a simple, efficient, high voltage radio frequency (RF) generator powered by a single voltage source (1.5-7 V) to resonantly drive ion traps or other capacitive loads. Our circuit is able to deliver RF voltages > 500 V(p-p) at frequencies ranging from 0.1 to 10 MHz. This RF oscillator uses low-cost, commercially available components, and can be easily assembled onto a circuit board of a few cm(2). Because of its simplicity and good efficiency, this circuit is useful in applications requiring small size and low power consumption such as portable ion trap systems where the duration of operation under battery power is of concern.

3.
FEBS Lett ; 428(1-2): 75-9, 1998 May 22.
Article in English | MEDLINE | ID: mdl-9645479

ABSTRACT

Two novel highly basic type 1 (single chain) ribosome-inactivating proteins (RIPs) with N-glycosidase activity have been found in elderberries (the fruits of Sambucus nigra L.). Mass spectrometry of these RIPs, which we named nigritins f1 and f2, gave Mr values of 24095 and 23 565, respectively. Both proteins strongly inhibited protein synthesis in rabbit reticulocyte lysates but were inactive against plant ribosomes. Both nigritins have a similar topological activity on pBlueScript SK+ DNA as that displayed by dianthin 30. Nigritin f1 is a constitutive RIP since it is present in both green and mature intact elderberries at nearly the same proportion with respect to total fruit protein. By contrast, nigritin f2 is inducible and only appeared in mature intact elderberries. Elderberries also contain two isoforms of a basic nigrin equivalent to the recently found basic nigrin b in elder bark (De Benito et al., FEBS Letters 413 (1997) 85-91). Our results indicate that probably not all plant RIPs exert the same biological function and that this may be determined by the physiological state of the tissue.


Subject(s)
Isoenzymes/metabolism , N-Glycosyl Hydrolases/metabolism , Plant Proteins/metabolism , Protein Synthesis Inhibitors/metabolism , Amino Acid Sequence , Animals , Isoenzymes/chemistry , Isoenzymes/isolation & purification , Molecular Sequence Data , N-Glycosyl Hydrolases/chemistry , N-Glycosyl Hydrolases/isolation & purification , Plant Proteins/chemistry , Plant Proteins/isolation & purification , Protein Synthesis Inhibitors/chemistry , Protein Synthesis Inhibitors/isolation & purification , Rabbits , Ribosome Inactivating Proteins , Ribosome Inactivating Proteins, Type 2
4.
Planta ; 204(3): 310-9, 1998 Mar.
Article in English | MEDLINE | ID: mdl-9530875

ABSTRACT

Mature leaves of dwarf elder (Sambucus ebulus L.) contain the non-toxic type 2 ribosome-inactivating protein ebulin 1 (Girbés et al., 1993b, J. Biol. Chem. 268: 18195-18199). We have now found that the green fruits of dwarf elder contain both free and polymerized forms of ebulin (ebulin f) and a new homodimeric D-galactose-binding lectin (SELfd). Polymerized material containing ebulin and lectin is composed of aggregates of variable relative molecular mass, some of them being close to 250,000. These aggregate forms are maintained in part by reducible disulphide bridges and reconstitute from reductant-free dialyzed material previously reduced with 2-mercaptoethanol. Direct incubation of free ebulin f with the free SELfd did not lead to polymerization, thus indicating that polymerization triggers some kind of substantial and perhaps catalyzed change in the structure of these proteins. Ebulin-containing polymerized material reacts with anti-ebulin f antibodies. Our results indicate that ebulin f is a fruit-form of ebulin 1. In contrast to green fruits, mature fruits lack both polymerized material and ebulin f, thus indicating some kind of reserve role for them in green fruits. Polymerization of ebulin and the dimeric lectin may represent a novel means of storing the non-toxic type 2 ribosome-inactivating proteins and lectins found in highly metabolic tissues, such as green fruits.


Subject(s)
Plant Proteins/chemistry , Trees/chemistry , Amino Acid Sequence , Biopolymers , Cross Reactions , Galactose/metabolism , Molecular Sequence Data , Plant Proteins/metabolism , Plant Proteins/pharmacology , Protein Binding , Protein Biosynthesis/drug effects , Ribosome Inactivating Proteins, Type 2 , Sequence Homology, Amino Acid
5.
FEBS Lett ; 413(1): 85-91, 1997 Aug 11.
Article in English | MEDLINE | ID: mdl-9287122

ABSTRACT

A novel, strongly basic, two-chain ribosome-inactivating protein (RIP) with an apparent Mr of 64000 by SDS-PAGE and 63469 by mass spectrometry analysis, that we have named basic nigrin b, has been found in the bark of elder (Sambucus nigra L.). The new protein does not agglutinate red blood cells, even at high concentrations and displays an unusually and extremely high activity towards animal ribosomes (IC50 of 18 pg/ml for translation by rabbit reticulocyte lysates). However, it is inactive against plant and HeLa cells protein synthesis. Our functional and structural data are consistent with a heterodimeric structure for basic nigrin b of the type A-B*, B* being a truncated lectin lacking functional binding domains equivalent to the B (lectin) chain of the type 2 RIP SNA I and nigrin b present also in elder bark.


Subject(s)
N-Glycosyl Hydrolases/chemistry , N-Glycosyl Hydrolases/isolation & purification , Plant Proteins/chemistry , Plant Proteins/isolation & purification , Trees/chemistry , Amino Acid Sequence , DNA Topoisomerases, Type I/metabolism , Dose-Response Relationship, Drug , HeLa Cells/drug effects , HeLa Cells/metabolism , Humans , N-Glycosyl Hydrolases/metabolism , N-Glycosyl Hydrolases/pharmacology , Plant Proteins/metabolism , Plant Proteins/pharmacology , Ribosome Inactivating Proteins , Ribosome Inactivating Proteins, Type 2 , Ribosomes/enzymology , Sequence Homology, Amino Acid
6.
Cell Mol Biol (Noisy-le-grand) ; 43(4): 485-99, 1997 Jun.
Article in English | MEDLINE | ID: mdl-9220142

ABSTRACT

A new N-glycosidase ribosome-inactivating protein (RIP) belonging to the novel family of the nontoxic type 2 RIPs from Sambucaceae has been isolated from rhizomes of dwarf elder (Sambucus ebulus L.) and named ebulin r. Dwarf elder rhizomes also contain a novel monomeric N-Ac-galactosamine-binding lectin that we named SEAII. Ebulin r and SEAII have two isoforms each one, which were readily resolved by ion exchange. Both isoforms of ebulin (ebulins r1 and r2) strongly inhibited protein synthesis in mammalian but not in plant ribosomes by promoting depurination of sensitive ribosomes. Ebulin r and SEAII have apparent molecular masses of 56 and 33.5 kDa, respectively. Ebulins r1 and r2 are composed of two dissimilar subunits (types A-B) of apparent molecular masses of 26 and 30 kDa by disulphide bridges. The rhizome SEAII and the lectins SNA II and SNA III from elder (Sambucus nigra L.) share good amino acid sequence homology. This rhizome ebulin-A chain is more sequence-related to RIP members of cucurbitaceae than to any other plant family. The rhizome ebulin B chain shares a large homology in amino acid sequence with ebulin 1-B chain and SEAII. Anti-ebulin 1 polyclonal antibodies raised in rabbits reacted better with ebulin r1 than with ebulin r2, thus suggesting that both RIP isoforms could have some differences.


Subject(s)
N-Glycosyl Hydrolases/metabolism , Plant Proteins/isolation & purification , Plants/metabolism , Amino Acid Sequence , Electrophoresis, Polyacrylamide Gel , Mass Spectrometry , Molecular Sequence Data , Ribosome Inactivating Proteins , Ribosome Inactivating Proteins, Type 2
7.
Arch Toxicol ; 71(6): 360-4, 1997.
Article in English | MEDLINE | ID: mdl-9195017

ABSTRACT

Nigrin b, a lectin isolated from the bark of elderberry (Sambucus nigra L.), has structure and enzymatic activity similar to that of ricin and other type 2 ribosome inactivating proteins (RIPs), and yet is much less toxic to cells and animals. In an attempt to explain this difference, we studied (1) the cytotoxicity of both lectins at 18 and 37 degrees C, and in the presence of substances interfering with intracellular routing, and (2) the binding of nigrin b to, and its uptake and degradation by HeLa cells, in parallel with ricin. As compared with the latter, (1) less nigrin b was bound and more was degraded by cells, with a resulting lower concentration remaining inside the cells, and (2) there is evidence for a different intracellular routing followed by the two lectins. These results may explain at least partly the different cytotoxicity and consequently the lower toxicity to mice of nigrin b compared with ricin.


Subject(s)
HeLa Cells/drug effects , N-Glycosyl Hydrolases/toxicity , Plant Proteins/toxicity , Ribosome Inactivating Proteins/toxicity , Ricin/toxicity , Analysis of Variance , Animals , Binding, Competitive , HeLa Cells/cytology , HeLa Cells/metabolism , Humans , Lethal Dose 50 , Mice , N-Glycosyl Hydrolases/isolation & purification , N-Glycosyl Hydrolases/metabolism , Plant Lectins , Plant Proteins/isolation & purification , Plant Proteins/metabolism , Protein Biosynthesis , Ribosome Inactivating Proteins/isolation & purification , Ribosome Inactivating Proteins/metabolism , Ribosome Inactivating Proteins, Type 2 , Ricin/metabolism , Temperature , Trees
8.
Cell Mol Biol (Noisy-le-grand) ; 42(4): 461-71, 1996 Jun.
Article in English | MEDLINE | ID: mdl-8828901

ABSTRACT

Plant ribosome-inactivating proteins (RIPs) are inhibitors present in all parts of plants that irreversibly inactivate eukaryotic ribosomes, thus impairing protein synthesis. RIPs are enzymes with N-glycosidase activity on the large rRNA. Their powerful inhibitory activity has been made use of advantageously to construct conjugates with suitable carriers targeted to altered specific cells. RIPs may be used to inhibit replication of both animal and plant viruses. The introduction of genes coding for RIPs into the genome of plants leads to an increase in resistance towards fungal pathogens and viruses. RIPs are important tools for the treatment of cancer and AIDS and for the protection of crop production.


Subject(s)
Antiviral Agents , Immunotoxins , Plant Proteins , Ribosomes , Antiviral Agents/metabolism , Antiviral Agents/therapeutic use , Forecasting , Immunotoxins/genetics , Immunotoxins/metabolism , Immunotoxins/therapeutic use , Plant Proteins/genetics , Plant Proteins/metabolism , Plant Proteins/therapeutic use , Plants, Genetically Modified
9.
Cell Mol Biol (Noisy-le-grand) ; 42(4): 473-6, 1996 Jun.
Article in English | MEDLINE | ID: mdl-8828902

ABSTRACT

The new type 2 RIPs ebulin l, nigrin b and nigrin f present in Sambucus display toxicity to HELA cells several orders of magnitude lower than that displayed by ricin. Despite N-terminal amino acid homology between the three RIPs in both the A and the B chains, these compounds display very different degrees of toxicity to HELA cells that does not seem to be paralleled by immunologic correlations. It is suggested that small changes in the protein structure are most probably responsible for the different degrees of toxicity.


Subject(s)
N-Glycosyl Hydrolases , Plant Proteins/toxicity , Protein Synthesis Inhibitors/toxicity , Ricin/toxicity , Cross Reactions , Enzyme-Linked Immunosorbent Assay , HeLa Cells , Humans , Ribosome Inactivating Proteins, Type 2
11.
FEBS Lett ; 360(3): 299-302, 1995 Mar 06.
Article in English | MEDLINE | ID: mdl-7883051

ABSTRACT

A new family of single chain (type 1) ribosome-inactivating proteins (RIPs), that we have named ebulitins, have been found in mature leaves of Sambucus ebulus L., a caprifoliaceae plant also known to contain a non-toxic two chain (type 2) RIP named ebulin I in its leaves. Ebulitins are basic proteins of M(r) 32,000, 29,000 and 29,000 for ebulitins alpha, beta and gamma, respectively. The simultaneous presence of different basic type 1 and acidic type 2 RIPs in the same plant and in the same tissue is described here for the first time and opens a new door in research into RIPs.


Subject(s)
Glycoside Hydrolases/isolation & purification , N-Glycosyl Hydrolases , Plant Proteins/isolation & purification , Protein Synthesis Inhibitors/isolation & purification , Amino Acids/analysis , Molecular Weight , Plant Proteins/chemistry , Plant Proteins/pharmacology , RNA, Ribosomal/metabolism , Ribosome Inactivating Proteins, Type 2 , Ribosomes/drug effects
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