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Conformational relaxation of a low-temperature protein as probed by photochemical hole burning. Horseradish peroxidase.

For the first time, conformational relaxation processes have been measured in a small protein, mesoporphyrin-horseradish peroxidase via their influence on spectral diffusion broadening of holes burnt in the fluorescence excitation spectrum of free base mesoporphyrin. Holes were burnt in three 0----0 bands of different tautomeric forms of the chromophore at 1.5 and 4 K, and the spectral diffusion broadening was measured in temperature cycling experiments between 4 and 30 K. The inhomogeneous linewidth for the tautomeric 0----0 bands was estimated to be 60-70 cm-1; the hole width was found narrow, being in the order of 350 MHz (10(-2) cm-1) at 1.5 K what allowed for an extremely sensitive detection of the conformational changes. Though proteins have many features in common with glasses, the spectral diffusion broadening of photochemical holes under temperature cycling conditions in mesoporphyrin horseradish peroxidase has a very different pattern as a function of temperature. Up to 12 K, the linewidth did not significantly change, then around 14 K; a steplike broadening was observed for all three tautomers, although to a different extent. The total magnitude of line broadening up to 30 K was large and also different for the tautomers. We argue that the difference between the behavior of this protein and that of glassy matrices originate from finite size effects; the protein may be characterized by a small number of TLS, and their distribution may bear discrete features.

Horseradish Peroxidase↗

Trauma care systems in India.

Trauma-care systems in India are at a nascent stage of development. Industrialised cities, rural towns and villages coexist, with almost complete lack of organised trauma care. There is gross disparity between trauma services available in various parts of the country. Rural India has inefficient services for trauma care, due to the varied topography, financial constraints and lack of appropriate health infrastructure. There is no national lead agency to co-ordinate various components of a trauma system. No mechanism for accreditation of trauma centres and professionals exists. Education in trauma life-support (TLS) skills has only recently become available. A nation-wide survey encompassing various facilities has demonstrated significant deficiencies in current trauma systems.Although injury is a major public-health problem, the Government of India has failed to recognise it as a priority. Significant efforts to develop trauma-care systems across the country are seen mainly in the private sector. New initiatives under National Health Policy 2002 are expected to result in improvement in the systems, but the allocation of funds remains grossly inadequate for any significant impact on the outcome.

Accidents, Traffic↗

The property of DNA polymerase zeta: REV7 is a putative protein involved in translesion DNA synthesis and cell cycle control.

Translesion DNA synthesis (TLS) is an important damage tolerance system which rescues cells from severe injuries caused by DNA damage. Specialized low fidelity DNA polymerases in this system synthesize DNA past lesions on the template DNA strand, that replicative DNA polymerases are usually unable to pass through. However, in compensation for cell survival, most polymerases in this system are potentially mutagenic and sometimes introduce mutations in the next generation. In yeast Saccharomyces cerevisiae (S. cerevisiae), DNA polymerase zeta, which consists of Rev3 and Rev7 proteins, and Rev1 are known to be involved in most damage-induced and spontaneous mutations. The human homologs of S. cerevisiae REV1, REV3, and REV7 were identified, and it is revealed that the human REV proteins have similar functions to their yeast counterparts, however, a large part of the mechanisms of mutagenesis employing REV proteins are still unclear. Recently, the new findings about REV proteins were reported, which showed that REV7 interacts not only with REV3 but also with REV1 in human and that REV7 is involved in cell cycle control in Xenopus. These findings give us a new point of view for further investigation about REV proteins. Recent studies of REV proteins are summarized and several points are discussed.

Animals↗

The proofreading 3'-->5' exonuclease activity of DNA polymerases: a kinetic barrier to translesion DNA synthesis.

The 3'-->5' exonuclease activity intrinsic to several DNA polymerases plays a primary role in genetic stability; it acts as a first line of defense in correcting DNA polymerase errors. A mismatched basepair at the primer terminus is the preferred substrate for the exonuclease activity over a correct basepair. The efficiency of the exonuclease as a proofreading activity for mispairs containing a DNA lesion varies, however, being dependent upon both the DNA polymerase/exonuclease and the type of DNA lesion. The exonuclease activities intrinsic to the T4 polymerase (family B) and DNA polymerase gamma (family A) proofread DNA mispairs opposite endogenous DNA lesions, including alkylation, oxidation, and abasic adducts. However, the exonuclease of the Klenow polymerase cannot discriminate between correct and incorrect bases opposite alkylation and oxidative lesions. DNA damage alters the dynamics of the intramolecular partitioning of DNA substrates between the 3'-->5' exonuclease and polymerase activities. Enzymatic idling at lesions occurs when an exonuclease activity efficiently removes the same base that is preferentially incorporated by the DNA polymerase activity. Thus, the exonuclease activity can also act as a kinetic barrier to translesion synthesis (TLS) by preventing the stable incorporation of bases opposite DNA lesions. Understanding the downstream consequences of exonuclease activity at DNA lesions is necessary for elucidating the mechanisms of translesion synthesis and damage-induced cytotoxicity.

DNA↗

European experience in the treatment of hyperuricemia.

The nonrecombinant form of urate oxidase has been routinely used in France since 1975 as standard practice in the initial management of non-Hodgkin's lymphoma (NHL) and acute lymphoblastic leukemia (ALL), and for prevention of tumor lysis syndrome (TLS). A retrospective study was performed to evaluate the frequency of metabolic complications and dialysis in 410 patients with B-cell stage III and IV NHL and L-3 ALL treated in France according to the LMB89 protocol, and to compare these results to those of other series of patients treated without urate oxidase. Of the 57 patients treated at Institut Gustave-Roussy, only five had metabolic complications occurring in the first cycle of chemotherapy. Two patients (3.5%) underwent dialysis: one because of oliguria, the second for preventive reasons. In all the other cases, metabolic problems were successfully resolved or prevented, using nonrecombinant urate oxidase (Uricozyme, Sanofi-Sythélabo, Inc, Paris, France) in combination with hyperhydration. Nonrecombinant urate oxidase is generally well tolerated. However, allergic reactions may occur, with rates varying from 0% to 4.5%. In addition, the extraction technology used to produce the product is limited by a low yield. A recombinant form of urate oxidase (rasburicase) was therefore developed. European clinical development results indicate that this agent produces a sharp and consistent decrease in uric acid levels in patients undergoing cytoreductive therapy. Additionally, there is a very low incidence of anaphylaxis. Studies have demonstrated the efficacy of urate oxidase in lowering uric acid levels, preventing hyperuricemia after the initiation of cytoreductive therapy, and preserving renal function in patients with B-cell advanced stage NHL and ALL.

Adolescent↗

Purification and characterization of Escherichia coli DNA polymerase V.

Cell survival and genome rescue after UV irradiation in Escherichia coli depends on DNA repair mechanisms induced in response to DNA damage as part of the SOS regulon. SOS occurs in two phases. The first phase is dominated by accurate repair processes such as excision and recombinational DNA repair, while the second phase is characterized by a large approximately 100-fold increase in mutations caused by an error-prone replication of damaged DNA templates. SOS mutagenesis occurs as a direct result of the action of the UmuDC gene-products, which form the low fidelity Escherichia coli DNA polymerase V, a heterotrimeric complex composed of UmuD'(2)C. This chapter describes the preparation of highly purified native pol V that is suitable for a wide range of biochemical studies of protein-protein, protein-DNA interactions and translesion-synthesis (TLS) mechanisms.

Adenosine Triphosphate↗

Genetic characterization of angiomatoid fibrous histiocytoma identifies fusion of the FUS and ATF-1 genes induced by a chromosomal translocation involving bands 12q13 and 16p11.

This case report documents the first karyotypic, fluorescence in situ hybridization, and genetic analysis of an angiomatoid fibrous histiocytoma that arose and recurred in the arm of a 5.5-year-old girl. Complex rearrangements between chromosomes 2, 12, 16, and 17 were noted, as well as deletion in the long arm of chromosome 11. Flow cytometry revealed a normal cell population. The t(12;16) site was further investigated using reverse transcriptase-polymerase chain reaction. We found that the FUS (also known as TLS) gene from 16p11 combined with the ATF-1 gene from 12q13 to generate a chimeric FUS/ATF-1. The FUS gene is rearranged in the t(12;16)(q13;p11) that characterizes myxoid liposarcoma and in acute myeloid leukemia with t(16;21)(p11;q22), while the ATF-1 gene is rearranged in the t(12;22)(q13;q12) found recurrently in clear cell sarcomas (malignant melanoma of soft parts). Thus, the FUS/ATF-1 gene in angiomatoid fibrous histiocytoma is predicted to code for a protein that is very similar to the chimeric EWS/ATF-1 found in clear cell sarcoma.

Activating Transcription Factor 1↗

Influence of additives on the protoplasts electrofusion.

Various neutral or charged surface active substances were used for testing the influence of additives on the electrofusion of barley protoplasts. It was found that neutral surface active agents DX, TAGB, Span-80 and AEO-9 could promote the electrofusion. The positively charged surface active agents Bardac 2080, Bardac 2280 and amphoteric surface active agents dodecyl-propyl betaine and CAB betaine also promote the electrofusion, but at high concentration the electrofusion efficiency will reduce. The negatively charged polymer agents Cibacron blue DX, Fluoresceinylthiocarbamoyl DX, and active surface substances K12 and Carsonol TLS- presented negative effect. These phenomena were discussed from the view of adsorption of additives on the membrane and the interactions between protoplasts.

Cell Fusion↗

Temporal lobe (TL) damage following surgery and high-dose photon and proton irradiation in 96 patients affected by chordomas and chondrosarcomas of the base of the skull.

PURPOSE: To determine the temporal lobe (TL) damage rate in 96 patients treated with high-dose proton and photon irradiation for chordomas and chondrosarcomas of the base of the skull. METHODS AND MATERIALS: The records of 96 consecutive patients treated at Massachusetts General Hospital (MGH) and Harvard Cyclotron Laboratory (HCL) between June 1984 and 1993, for chordomas and chondrosarcomas of the base of the skull were reviewed. All the patients had undergone some degree of resection of the tumor prior to radiation therapy. Seventy-five patients were classified as "primary tumors" and 21 as recurrent or regrowing tumors after one or more surgical procedures. All the patients were randomized to receive 66.6 or 72 cobalt Gray equivalent (CGE) on a prospective dose-searching study by proton and photon irradiation (Radiation Therapy Oncology Group #85-26) with conventional fractionation (1.8 CGE/day, 5 fractions/week). All treatments were planned using the three-dimensional (3D) planning system developed at the Massachusetts General Hospital, and the dose was delivered using opposed lateral fields for the photon component and a noncoplanar isocentric technique for the proton component. Clinical symptoms of TL damage were classified into 4 grades. Computerized tomography (CT) and magnetic resonance imaging (MRI) scans were evaluated for white matter changes. Abnormalities associated with persistent or recurrent tumor were distinguished from radiation-induced changes. TLs were delineated on the original scans of the 10 patients with damage and those of a group of 33 patients with no clinical or MRI evidence of injury. Dose distributions were calculated and dose-volume histograms were obtained for these patients. RESULTS: Of the patients, 10 developed TL damage, with bilateral injury in 2 and unilateral injury in 8. The cumulative TL damage incidence at 2 and 5 years was 7.6 and 13.2%, respectively. The MRI areas suggestive of TL damage were always separated from the tumor bed. Symptoms were severe to moderate in 8 patients. Several baseline factors, tumor- or host-related, were analyzed to evaluate their predictivity for TL damage: age, gender, tumor site, histology, type of presentation, type and number of surgical procedures, primary tumor volume, prescribed dose, normal tissue involvement, and volume of TL receiving doses ranging between 10 and 50 CGE or more. Only gender, in a univariate analysis (log rank) was a significant predictor of damage (0.0155), with male patients being at significantly higher risk of TL injury. In a stepwise Cox regression that included gender as a variable, no other baseline variable improved the prediction of damage. CONCLUSIONS: The 2- and 5-year cumulative TL damage rates were 7.6 and 13.2%, respectively. Despite the different TL damage rates related to age, tumor volume, number of surgical procedures prior to radiation therapy, and prescribed doses to the tumor, only gender was a significant predictor of damage (p = 0.0155) using a univariate (log rank) test. Chordomas and chondrosarcomas of the base of the skull may represent an interesting model to evaluate the TL damage rates because of their extradural origin, displacing the white matter instead of infiltrating it as gliomas do, because of their longer local recurrence-free survival other than gliomas and other brain tumors and because of the high doses of irradiation delivered to the target volume to obtain local control.

Adolescent↗

[Electro-clinical semiology of subintrant temporal lobe seizures].

The authors recorded 10 to 67 TLS (mean 29) in 10 patients (5 M, 5 F; 9 to 41 years, mean 23) during 'acute' (4-6 hours) stereo-EEG exploration. At the onset of seizures, we observed: subjective manifestations (often epigastric), autonomic symptoms, and oro-alimentary 'automatisms'. Affective, unpleasant, manifestations occurred in only 2 patients. The discharges may only affect the Ammon's horn, but they never involve only the amygdala. There is a strong relationship between the duration of the discharge, the number of cerebral structures (temporal and, later, extratemporal) involved in the discharge, and the clinical symptomatology. Somatomotor lateralized manifestations, and gestural 'automatisms' only occur when the discharges spread to extratemporal cerebral structures. The secondary generalizations are very rare and are also linked to a large extratemporal spread of the discharge.

Adolescent↗

Genomic and functional characterization of the oas gene family encoding O-acetylserine (thiol) lyases, enzymes catalyzing the final step in cysteine biosynthesis in Arabidopsis thaliana.

The final step of cysteine biosynthesis in plants is catalyzed by O-acetylserine (thiol) lyase (OAS-TL), which occurs as several isoforms found in the cytosol, the plastids and the mitochondria. Genomic DNA blot hybridization and isolation of genomic clones indicate single copy genes (oasA1, oasA2, oasB and oasC) that encode the activities of OAS-TL A, B and C found in separate subcellular compartments in the model plant Arabidopsis thaliana. Sequence analysis reveals that the newly discovered oasA2 gene represents a pseudogene that is still transcribed, but is not functionally translated. The comparison of gene structures suggests that oasA1/oasA2 and oasB/oasC are closely related and may be derived from a common ancestor by subsequent duplications. OAS-TL A, B and C were overexpressed in an Escherichia coli mutant lacking cysteine synthesis and exhibited bifunctional OAS-TL and beta-cyanoalanine synthase (CAS) activities. However, all three proteins represent true OAS-TLs according to kinetic analysis and are unlikely to function in cyanide detoxification or secondary metabolism. In addition, it was demonstrated that the mitochondrial OAS-TL C exhibits in vivo protein-protein interaction capabilities with respect to cysteine synthase complex formation similar to cytosolic OAS-TL A and plastid OAS-TL B. Multiple database accessions for each of the A. thaliana OAS-TL isoforms can thus be attributed to a specified number of oas genes to which functionally defined gene products are assigned, and which are responsible for compartment-specific cysteine synthesis.

Arabidopsis↗

Characterization and expression of the human gene encoding two translocation liposarcoma protein-associated serine-arginine (TASR) proteins.

Translocation liposarcoma protein (TLS)-associated serine-arginine (TASR)-1 and -2 are two newly identified serine-arginine splicing factors. Our recent studies suggest that disruption of TASR-mediated pre-mRNA splicing is involved in the pathogenesis of human leukemia and sarcomas. The mRNA transcripts for TASR-1 and -2 share an identical sequence at the 5' untranslated region (5' UTR) and in part of the coding region; however the other regions of the transcripts diverge from each other and it was not clear whether the differences resulted from alternative splicing or transcription from two distinct genes. Here we describe the assignment of both TASR cDNAs to the same 16 kb DNA segment located on chromosome 1. Despite the presence of at least three retroposed products of TASR-1 mRNA in the human genome, only the 16 kb structural TASR gene on chromosome 1 is actively transcribed. In addition, multiple polyadenylation sites and a rare U12-type intron were found within the TASR gene. Transcription initiation site of the TASR gene was determined by primer extension; analysis of the TASR promoter revealed that it lacks the TATA box but contains a GC-rich sequence. When cloned into a luciferase reporter and transfected into human cells, the TASR promoter construct generated luciferase activity that was at least 2000 fold greater than the promoterless plasmid. Northern blot analysis showed that at least five different TASR-1 and -2 transcripts are expressed in a broad range of human tissues.

Alternative Splicing↗

Reduced sensitivity to and ras mutation spectrum of N-ethyl-N-nitrosourea-induced thymic lymphomas in adult C.B-17 scid mice.

Scid mice are defective in the ability to repair DNA double strand breaks and, as a consequence, their cells are radiosensitive. Further, they have been shown to be prone to develop thymic lymphomas (TLs) after small doses of ionizing radiation. Little is known, however, on the role of scid mutation in chemical carcinogenesis. To determine if scid mutation increased predisposition to chemical carcinogenesis, we examined both the susceptibility of scid mice to N-ethyl-N-nitrosourea (ENU)-induced lymphomagenesis and the involvement of ras gene activation. Adult female mice at 8 weeks of age were given ENU in their drinking water at 400 ppm for 2-10 weeks. Contrary to expectations, we observed a two to three-fold reduction in TL development in the scid mice. The highest incidence was achieved by ENU treatment for 8 weeks for scid and wild-type C.B-17 mice, of 42 and 85%, respectively (P<0.05). We investigated whether this was attributable to the usage of the ras mutation pathway. There was, however, no significant difference in the frequency and spectrum of K-ras mutation between the scid and wild-type C.B-17 mice. Most of the K-ras mutations were either GGT to GAT transition in codon 12 (11/23: 48%) or CAA to CCA transversion in codon 61 (8/23: 35%) that was independent of scid background. The incidence of N-ras mutation was very low. These results indicate that scid mice are less susceptible to ENU-induced lymphomagenesis and ras gene mutation frequently occurs in both scid and wild-type C.B-17 mice.

Animals↗

Functions of human DNA polymerases eta, kappa and iota suggested by their properties, including fidelity with undamaged DNA templates.

Human DNA polymerases eta, kappa and iota are template-dependent, Y-family DNA polymerases that have been implicated in translesion DNA synthesis (TLS) in human cells. Here, we briefly review evidence that these exonuclease-deficient polymerases copy undamaged DNA with very low fidelity and unusual error specificity. Based on the base substitution specificity and other biochemical properties of DNA polymerases eta and iota, we consider the possibility that they participate in specialized DNA transactions that repair damaged DNA and/or generate mutations in the variable regions of immunoglobulin genes.

DNA↗

Polymorphism of genes encoding SOS polymerases in natural populations of Escherichia coli.

High fidelity replicative DNA polymerases can be blocked during DNA replication by various base damages, which represents a potentially lethal event. Escherichia coli possesses three DNA polymerases, PolII, PolIV and PolV, that can continue replication over such lesions in template DNA, thus allowing for cell survival. Genes coding for these enzymes, polB, dinB, and umuCD respectively, belong to the stress-inducible SOS regulon. We have analyzed the patterns of nucleotide sequence variability of genes encoding for three SOS polymerases from E. coli natural isolates in order to identify the nature of selective forces that determine their evolution. The frequency of inferred inter-strain recombination events, and the frequency of synonymous and non-synonymous base substitutions within these genes do not deviate significantly from those observed for the control group composed of 2 genes coding for DNA polymerases PolI and PolIII and 10 metabolic genes. This suggests that the loci coding for SOS polymerases are subject to selective pressure for the maintenance of their function and specificity. The fact that genes coding for translesion-synthesis (TLS) polymerases, particularly dinB and umuC homologs, have been conserved during evolution and the present analysis suggest that their activity is essential for the cellular survival and fitness.

Bacterial Proteins↗

Multifunctional zinc finger proteins in development and disease.

Post-transcriptional processes contribute significantly towards the generation of proteomic diversity. An increasing number of mutations have been described that affect genes encoding components of the post-transcriptional machinery. In particular, multifunctional proteins that link transcription with post-transcriptional processes have been implicated in several human diseases including cancer. A predominant feature of these proteins is the zinc finger, an ancient structural motif that mediates protein ratio protein interactions and is capable of interacting with both DNA and RNA. Zinc finger proteins are the most abundant class of proteins in the human proteome, yet the majority remain uncharacterised. Here we describe multifunctional zinc finger proteins linked to human development and disease. The examples discussed are WT1, ZNF74, EWS, TLS, TAFII68, YY1, CTCF and the GLI proteins. The study of these and other zinc finger proteins provides insights into the functional versatility of the zinc finger motif and suggests that both alternative splicing and sub-cellular compartmentalisation may modulate their multifunctionality.

DNA↗

Order theoretical tools for the evaluation of complex regional pollution patterns.

The mathematical and statistical evaluation of environmental data gains an increasing importance in environmental chemistry as the data sets become more complex. It is inarguable that different mathematical and statistical methods should be applied in order to compare results and to enhance the possible interpretation of the data. Very often several aspects have to be considered simultaneously, for example, several chemicals entailing a data matrix with objects (rows) and variables (columns). In this paper a data set is given concerning the pollution of 58 regions in the state of Baden-Württemberg, Germany, which are polluted with metals lead, cadmium, zinc, and with sulfur. For pragmatic reasons the evaluation is performed with the dichotomized data matrix. First this dichotomized 58 x 13 data matrix is evaluated by the Hasse diagram technique, a multicriteria evaluation method which has its scientific origin in Discrete Mathematics. Then the Partially Ordered Scalogram Analysis with Coordinates (POSAC) method is applied. It reduces the data matrix in plotting it in a two-dimensional space. A small given percentage of information is lost in this method. Important priority objects, like maximal and minimal objects (high and low polluted regions), can easily be detected by Hasse diagram technique and POSAC. Two variables attained exceptional importance by the data analysis shown here: TLS, Sulfur found in Tree Layer, is difficult to interpret and needs further investigations, whereas LRPB, Lead in Lumbricus Rubellus, seems to be a satisfying result because the earthworm is commonly discussed in the ecotoxicological literature as a specific and highly sensitive bioindicator.

Analysis of Variance↗

Synthesis, Properties, and Crystal Structures of Benzene-1,2-dithiolato Complexes of Thallium(I) and -(III).

The syntheses, molecular structures and properties of homoleptic 1,2-S(2)C(6)H(4) complexes of thallium(I) and thallium(III) with four-coordinated metal centers are described. Anaerobic treatment of TlCl, TlNO(3), or Tl(2)CO(3) with solutions of sodium methanolate and 1,2-(HS)(2)C(6)H(4) in methanol gave after metathesis with [NEt(4)]Br yellow solutions of [NEt(4)](2)[{Tl(1,2-(&mgr;-S)(2)C(6)H(4))}(2)] ([NEt(4)](2)1). Yellow single crystals were obtained from saturated acetone solutions at -10 degrees C and the crystal data for [NEt(4)](2)1 are monoclinic, P2(1)/c, with Z = 2, a = 7.440(1) Å, b = 16.373(3) Å, c = 13.201(2) Å, and beta = 97.08(1) degrees. Complex 1(2)(-)(), the first structurally characterized homoleptic ionic thiolate complex of thallium(I), contains rectangular bipyramidal [TlS(4)Tl] cages with the four sulfur atoms defining the equatorial plane and the two thallium atoms in axial positions. The S(2)C(6)H(4) fragments are almost coplanar with the S(4) plane. In the crystal lattice, nearly linear Tl.Tl chains align along the a-axis (offset ca. 3.0 degrees ) with the ligand planes parallel to the bc-plane. Within and between dimers short Tl.Tl distances are observed (Tl.Tl' within a dimeric unit, 3.5116(4) Å; Tl.Tl between dimeric units, 3.9371(3) Å) with the distance between dimeric units being the shortest contact between anions-Tl.S distances between dimeric units are longer than 5.8 Å. Aerobic treatment of TlCl, TlNO(3), or Tl(2)CO(3) with solutions of sodium methanolate and 1,2-(HS)(2)C(6)H(4) in methanol and metathesis with [NEt(4)]Br led to [NEt(4)][Tl(1,2-S(2)C(6)H(4))(2)] ([NEt(4)]2). Yellow single crystals were obtained from saturated acetone solutions at 0 degrees C and the crystal data for [NEt(4)]2 are orthorhombic, Pnn2, with Z = 2, a = 11.449(2) Å, b = 10.060(2) Å, c = 9.950(2) Å. Complex 2(-) is the first homoleptic four-coordinate thiolate of thallium(III) and contains the unusually short Tl-S distance of 2.469(4) Å. In solution, ion pairing results in chemical and magnetic inequivalence of the S(2)C(6)H(4) ligands. Although both preparations employ the reaction of thallium(I) salts with 1,2-(NaS)(2)C(6)H(4) in a 1:2 stoichiometry, complex 1(2)(-) is probably not an intermediate to the formation of 2(-). Exposing anaerobically prepared solutions of 1(2)(-) to air results in a series of color changes in the solution over a 20 min period; however, 2(-) could not be observed by NMR spectroscopy.

Journal Article↗