Telomerase Activity Is Sensitive to Subtle Perturbations of the TLC1 Pseudoknot 3′ Stem and Tertiary Structure

Pseudoknot formation in the core region of the telomerase RNA has been demonstrated to be important for telomerase activity in vertebrates, ciliates, and yeast. Characterization of the Saccharomyces cerevisiae telomerase RNA (TLC1) pseudoknot identified tertiary structural interactions that are also important for telomerase activity, as previously observed for the Kluyveromyces lactis and human telomerase RNA pseudoknots. In addition, the contributions of backbone ribose 2′-OH groups in the pseudoknot to telomerase catalysis were investigated previously, using 2′-OH (ribose) to 2′-H (deoxyribose) or 2′-O-methyl substitutions in the stem 2 helix, and it was proposed that one or more 2′-OH groups from the stem 2 sequences at or near the triple helix participate in telomerase catalysis. Based on these studies and investigations of the structural and thermodynamic properties of the TLC1 RNA pseudoknot region, we have examined the structural and thermodynamic perturbations of the 2′-O-methyl and 2′-H substituted pseudoknots, using UV-monitored thermal denaturation, native gel electrophoresis, and circular dichroism spectroscopy. Our results demonstrate the presence of A-form helical geometry perturbations in the backbone sugar substituted pseudoknots, show a correlation between thermodynamic stability and telomerase activity, and are consistent with the identification of the U809 ribose 2′-OH as a potential contributor to telomerase activity.     

Creation of salt-insensitive 3'(2'),5'-bisphosphate nucleotidase by modeling and mutagenesis approach

3′(2′),5′-Bisphosphate nucleotidase, (EC 3.1.3.7) (BPntase) is a ubiquitous enzyme. Recently, these enzymes have drawn considerable attention as in vivo targets of salt toxicity as well as therapeutic targets of lithium that is used for the treatment of manic-depressive disorders. They belong to the Mg²⁺-dependent Li⁺-sensitive phosphomonoesterase super-family and are highly sensitive to lithium and sodium ions. However, the molecular mechanism of inhibition of this group of enzymes by monovalent cations has not been completely understood. Previously we have identified a BPntase (Dhal2p) from a highly halotolerant yeast Debaryomyces hansenii. Molecular characterization revealed a number of unique features in Dhal2p, indicating this is an extraordinary member of the family. In this study, we have carried out the structure-function analysis of Dhal2p through the combination of molecular modeling and in vitro mutagenesis approach. We have not only provided the explanation for the role played by the functionally important elements that are conserved among the members of this family but also identified important, novel structural elements in this enzyme. Our study for the first time unraveled the role of a flap as well as a loop region in the functioning of this enzyme. Most importantly, mutations in the loop region resulted in the creation of a BPntase that was insensitive to salt.     

4,4'-diisothiocyano-2 ,2'-stilbenedisulfonate protects cultured cerebellar granule neurons from death

We examined the effects of 4,4′-diisothiocyano-2,2′-stilbenedisulfonate (DIDS), an inhibitor of the chloride-bicarbonate exchangers and chloride channels, on death in cultured cerebellar granule neurons. Various stimuli, such as reduction of extracellular K⁺ concentration, removal of growth factors, and staurosporine treatment, induced cell death. This death was blocked by DIDS in a dose dependent manner. In the presence of DIDS, the cells exposed to such stimuli did not show DNA fragmentation, but retained the ability to exclude trypan blue and to metabolize MTT to formazan. On the other hand, pretreatment of the cells with DIDS did not show any protective effects. The neuroprotective effect of DIDS was not influenced by extracellular Na⁺, Cl⁻, HCO₃⁻ or Ca²⁺ concentrations, although reduction of extracellular Cl⁻ or Ca²⁺ concentrations per se induced neuronal death. Other chloride-bicarbonate exchange blockers like 4-acetamido-4′-isothiocyanatostilmene-2,2′-disulfonic acid (SITS) or 4,4′-dinitrostilbene-2,2′-disulfonic acid (DNDS) showed no significant effects on neuronal survival under these death-inducing stimuli. Dimethylamiloride, an inhibitor of the Na⁺/H⁺ exchanger, did not influence neuronal death induced by these stimuli. Cells undergoing death showed gradual intracellular acidification, and DIDS did not inhibit this response, although DIDS (2 mM) per se induced transitory acidification followed by recovery within 10 min. DIDS did not influence intracellular Ca²⁺ or Cl⁻ levels during the lethal process. DIDS suppressed the cleavage of caspase-3 in the cells exposed to the death-inducing stimuli. These findings suggest that the neuroprotective effect of DIDS is mediated by a novel mechanism other than by nonselective inhibition of transporters or channels, and that DIDS blocks the death program upstream of caspases and downstream of all of the activation processes triggered by various stimuli.     

Biochanin-A ameliorates pulmonary fibrosis by suppressing the TGF-β mediated EMT,myofibroblasts differentiation and collagen deposition in in vitro and in vivo systems

Background: Idiopathic Pulmonary Fibrosis (IPF) is a progressive inflammatory disorder driven by a fibrotic cascade of events such as epithelial to mesenchymal transition, extracellular matrix production and collagen formation in the lungs in a sequential manner. IPF incidences were raising rapidly across the world. FDA approved pirfenidone and nintedanib (tyrosine kinase inhibitors) are being used as a first-line treatment drugs for IPF, however, neither the quality of life nor survival rates have been improved because of patient noncompliance due to multiple side effects. Thus, the development of novel therapeutic approaches targeting TGF-β mediated cascade of fibrotic events is urgently needed to improve the survival of the patients suffering from devastating disease.Purpose: The aim of this study was to investigate and validate the anti-fibrotic properties of Biochanin-A (isoflavone) against TGF-β mediated fibrosis in in vitro, ex vivo, in vivo models and to determine the molecular mechanisms that mediate these anti-fibrotic effects.Methods: The therapeutic activity of BCA was determined in in vitro/ex vivo models. Cells were pre-treated with BCA and incubated in presence or absence of recombinant-TGF-β to stimulate the fibrotic cascade of events. Pulmonary fibrosis was developed by intratracheal administration of bleomycin in rats. BCA treatment was given for 14 days from post bleomycin instillation and then various investigations (collagen content, fibrosis gene/protein expression and histopathological changes) were performed to assess the anti-fibrotic activity of BCA.Results: In vitro/ex vivo (Primary normal, IPF cell line and primary IPF cells/ Precision cut mouse lung slices) experiments revealed that, BCA treatment significantly (p < 0.001) reduced the expression of TGF-β modulated fibrotic genes/protein expressions (including their functions) which are involved in the cascade of fibrotic events. BCA treatment significantly (p < 0.01) reduced the bleomycin-induced inflammatory cell-infiltration, inflammatory markers expression, collagen deposition and expression of fibrotic markers in lung tissues equivalent or better than pirfenidone treatment. In addition, BCA treatment significantly (p < 0.001) attenuated the TGF-β1/BLM-mediated increase of TGF-β/Smad2/3 phosphorylation and resulted in the reduction of pathological abnormalities in lung tissues determined by histopathology observations.Conclusion: Collectively, BCA treatment demonstrated the remarkable therapeutic effects on TGF-β/BLM mediated pulmonary fibrosis using IPF cells and rodent models. This current study may offer a novel treatment approach to halt and may be even rescue the devastating lung scarring of IPF.     

An analysis of temperature adaptation in cold active, mesophilic and thermophilic Bacillus α-amylases

A comparative biochemical and structural study was performed on a cold active α-amylase from Bacillus cereus (BCA) and two well-known homologous mesophilic and thermophilic α-amylases from Bacillus amyloliquefaciens (BAA) and Bacillus licheniformis (BLA). In spite of a high degree of sequence and structural similarity, drastic variations were found for T_opt as 50, 70 and 90 °C for BCA, BAA and BLA, respectively. The half-lives of thermoinactivation were 1 and 9 min for BCA and BAA at 80 °C respectively, whilst there was no inactivation for BLA at this temperature. Thermodynamic studies on inactivation process suggested that lower thermostability of BCA is due to lower inactivation slope of the Arrhenius plots and subsequently, lower E_a and ΔH^#. Increased K_m and accessible surface area for catalytic residues along with a decreased number of internal interactions in this region in BCA compared to BLA suggest that BCA substrate-binding site might be temperature sensitive and is probably more flexible. On the other hand, fewer ion pairs, destructive substitutions and disruption of aromatic interaction networks in structurally critical regions of Bacillus α-amylases result in a severe decrease in BCA thermostability compared to its mesophilic and thermophilic homologues.     

Hepatic stellate cells are an important cellular site for β-carotene conversion to retinoid

Hepatic stellate cells (HSCs) are responsible for storing 90-95% of the retinoid present in the liver. These cells have been reported in the literature also to accumulate dietary β-carotene, but the ability of HSCs to metabolize β-carotene in situ has not been explored. To gain understanding of this, we investigated whether β-carotene-15,15′-monooxygensase (Bcmo1) and β-carotene-9′,10′-monooxygenase (Bcmo2) are expressed in HSCs. Using primary HSCs and hepatocytes purified from wild type and Bcmo1-deficient mice, we establish that Bcmo1 is highly expressed in HSCs; whereas Bcmo2 is expressed primarily in hepatocytes. We also confirmed that HSCs are an important cellular site within the liver for accumulation of dietary β-carotene. Bcmo2 expression was found to be significantly elevated for livers and hepatocytes isolated from Bcmo1-deficient compared to wild type mice. This elevation in Bcmo2 expression was accompanied by a statistically significant increase in hepatic apo-12′-carotenal levels of Bcmo1-deficient mice. Although apo-10′-carotenal, like apo-12′-carotenal, was readily detectable in livers and serum from both wild type and Bcmo1-deficient mice, we were unable to detect either apo-8′- or apo-14′-carotenals in livers or serum from the two strains. We further observed that hepatic triglyceride levels were significantly elevated in livers of Bcmo1-deficient mice fed a β-carotene-containing diet compared to mice receiving no β-carotene. Collectively, our data establish that HSCs are an important cellular site for β-carotene accumulation and metabolism within the liver.     

Induction of Tumor Cell Death through Targeting Tubulin and Evoking Dysregulation of Cell Cycle Regulatory Proteins by Multifunctional Cinnamaldehydes

Multifunctional trans-cinnamaldehyde (CA) and its analogs display anti-cancer properties, with 2-benzoyloxycinnamaldehyde (BCA) and 5-fluoro-2-hydroxycinnamaldehyde (FHCA) being identified as the ortho-substituted analogs that possess potent anti-tumor activities. In this study, BCA, FHCA and a novel analog 5-fluoro-2-benzoyloxycinnamaldehyde (FBCA), were demonstrated to decrease growth and colony formation of human colon-derived HCT 116 and mammary-derived MCF-7 carcinoma cells under non-adhesive conditions. The 2-benzoyloxy and 5-fluoro substituents rendered FBCA more potent than BCA and equipotent to FHCA. The cellular events by which these cinnamaldehydes caused G₂/M phase arrest and halted proliferation of HCT 116 cells were thereby investigated. Lack of significant accumulation of mitosis marker phospho-histone H3 in cinnamaldehyde-treated cells indicated that the analogs arrested cells in G₂ phase. G₂ arrest was brought about partly by cinnamaldehyde-mediated depletion of cell cycle proteins involved in regulating G₂ to M transition and spindle assembly, namely cdk1, cdc25C, mad2, cdc20 and survivin. Cyclin B1 levels were found to be increased, which in the absence of active cdk1, would fail to drive cells into M phase. Concentrations of cinnamaldehydes that brought about dysregulation of levels of cell cycle proteins also caused tubulin aggregation, as evident from immunodetection of dose-dependent tubulin accumulation in the insoluble cell lysate fractions. In a cell-free system, reduced biotin-conjugated iodoacetamide (BIAM) labeling of tubulin protein pretreated with cinnamaldehydes was indicative of drug interaction with the sulfhydryl groups in tubulin. In conclusion, cinnamaldehydes treatment at proapoptotic concentrations caused tubulin aggregation and dysegulation of cell cycle regulatory proteins cdk1 and cdc25C that contributed at least in part to arresting cells at G₂ phase, resulting in apoptotic cell death characterized by emergence of cleaved forms of caspase 3 and poly (ADP-ribose) polymerase (PARP). Results presented in this study have thus provided further insights into the intricate network of cellular events by which cinnamaldehydes induce tumor cell death.     

Prostasin may contribute to chemoresistance,repress cancer cells in ovarian cancer,and is involved in the signaling pathways of CASP/PAK2-p34/actin

Ovarian cancer is the deadliest of gynecologic cancers, largely due to the development of drug resistance in chemotherapy. Prostasin may have an essential role in the oncogenesis. In this study, we show that prostasin is decreased in an ovarian cancer drug-resistant cell line and in ovarian cancer patients with high levels of excision repair cross-complementing 1, a marker for chemoresistance. Our cell cultural model investigation demonstrates prostasin has important roles in the development of drug resistance and cancer cell survival. Forced overexpression of prostasin in ovarian cancer cells greatly induces cell death (resulting in 99% cell death in a drug-resistant cell line and 100% cell death in other tested cell lines). In addition, the surviving cells grow at a much lower rate compared with non-overexpressed cells. In vivo studies indicate that forced overexpression of prostasin in drug-resistant cells greatly inhibits the growth of tumors and may partially reverse drug resistance. Our investigation of the molecular mechanisms suggests that prostasin may repress cancer cells and/or contribute to chemoresistance by modulating the CASP/P21-activated protein kinase (PAK2)-p34 pathway, and thereafter PAK2-p34/JNK/c-jun and PAK2-p34/mlck/actin signaling pathways. Thus, we introduce prostain as a potential target for treating/repressing some ovarian tumors and have begun to identify their relevant molecular targets in specific signaling pathways.     

Black carrot anthocyanins exhibit neuroprotective effects against MPP+ induced cell death and cytotoxicity via inhibition of oxidative stress mediated apoptosis

Parkinson’s disease (PD) is a common chronic neurodegenerative disease induced by the death of dopaminergic neurons. Anthocyanins are naturally found antioxidants and well-known for their preventive effects in neurodegenerative disorders. Black carrots (Daucus carota L. ssp. sativus var. atrorubens Alef.) are a rich source of anthocyanins predominantly including acylated cyanidin-based derivatives making them more stable. However, there have been no reports analysing the neuroprotective role of black carrot anthocyanins (BCA) on PD. In order to investigate the potential neuroprotective effect of BCA, human SH-SY5Y cells were treated with MPP+?(1-methyl-4-phenylpyridinium) to induce PD associated cell death and cytotoxicity. Anthocyanins were extracted from black carrots and the composition was determined by HPLC–DAD. SH-SY5Y cells were co-incubated with BCA (2.5, 5, 10, 25, 50, 100 µg/ml) and 0.5 mM MPP+?to determine the neuroprotective effect of BCA against MPP+?induced cell death and cytotoxicity. Results indicate that BCA concentrations did not have any adverse effect on cell viability. BCA revealed its cytoprotective effect, especially at higher concentrations (50, 100 µg/ml) by increasing metabolic activity and decreasing membrane damage. BCA exhibited antioxidant activity via scavenging MPP+?induced reactive oxygen species (ROS) and protecting dopaminergic neurons from ROS mediated apoptosis. These results suggest a neuroprotective effect of BCA due to its high antioxidant and antiapoptotic activity, along with the absence of cytotoxicity. The elevated stability of BCA together with potential neuroprotective effects may shed light to future studies in order to elucidate the mechanism and further neuro-therapeutic potential of BCA which is promising as a neuroprotective agent.

     

2′,3′-cAMP hydrolysis by metal-dependent phosphodiesterases containing DHH, EAL, and HD domains is non-specific: Implications for PDE screening

The recent report of 2′,3′-cAMP isolated from rat kidney is the first proof of its biological existence, which revived interest in this mysterious molecule. 2′,3′-cAMP serves as an extracellular adenosine source, but how it is degraded remains unclear. Here, we report that 2′,3′-cAMP can be hydrolyzed by six phosphodiesterases containing three different families of hydrolytic domains, generating invariably 3′-AMP but not 2′-AMP. The catalytic efficiency (k_cat/K_m) of each enzyme against 2′,3′-cAMP correlates with that against the widely used non-specific substrate bis(p-nitrophenyl)phosphate (bis-pNPP), indicating that 2′,3′-cAMP is a previously unknown non-specific substrate for PDEs. Furthermore, we show that the exclusive formation of 3′-AMP is due to the P-O2′ bond having lower activation energy and is not the result of steric exclusion at enzyme active site. Our analysis provides mechanistic basis to dissect protein function when 2′,3′-cAMP hydrolysis is observed.     

1,4-Dioxane-2,5-dione-type monomers derived from l-ascorbic and d-isoascorbic acids. Synthesis and polymerisation

l-Ascorbic and d-isoascorbic acids have been used as the starting materials for the preparation of (3R,4′S)-3-(2′,2′-dimethyl-1′,3′-dioxolan-4′-yl)-1,4-dioxane-2,5-dione (IPTA), (3R and S, 4′S,6R)-3-methyl-6-(2′,2′-dimethyl-1′,3′-dioxolan-4′-yl)-1,4-dioxane-2,5-dione (IPTP) and (3R,4′R)-3-(2′,2′-dimethyl-1′,3′-dioxolan-4′-yl)-1,4-dioxane-2,5-dione (IPEA), three novel 1,4-dioxane-2,5-dione-type monomers. Ring-opening homopolymerisation and copolymerisation of the IPTA monomer, derived from l-ascorbic acid, with d,l-lactide have been performed. The polymers were characterised by elemental microanalysis, as well as IR and ¹H and ¹³C NMR spectroscopies. GPC was used to estimate product molecular weights, and thermal studies (DSC and TGA) revealed that all the polymers were amorphous, being stable up to 250 °C under nitrogen.     

Studying aminoglycoside modification by the acetyltransferase class of resistance-causing enzymes via microarray

Aminoglycosides are broad-spectrum antibacterials to which some bacteria have acquired resistance. The most common mode of resistance to aminoglycosides is enzymatic modification of the drug by different classes of enzymes including acetyltransferases (AACs). Thus, the modification of aminoglycosides by AAC(2′) from Mycobacterium tuberculosis and AAC(3) from Escherichia coli was studied using aminoglycoside microarrays. Results show that both enzymes modify their substrates displayed on an array surface in a manner that mimics their relative levels of modification in solution. Because aminoglycosides that are modified by resistance-causing enzymes have reduced affinities for binding their therapeutic target, the bacterial rRNA aminoacyl-tRNA site (A-site), arrays were probed for binding to a fluorescently labeled oligonucleotide mimic of the A-site after modification. A decrease in binding was observed when aminoglycosides were modified by AAC(3). In contrast, a decrease in binding of the A-site is not observed when aminoglycosides are modified by AAC(2′). Interestingly, these effects mirror the biological functions of the enzymes: the AAC(3) used in this study is known to confer aminoglycoside resistance, while the AAC(2′) is chromosomally encoded and unlikely to play a role in resistance. These studies lay a direct foundation for studying resistance to aminoglycosides and can also have more broad applications in identifying and studying non-aminoglycoside carbohydrates or proteins as substrates for acetyltransferase enzymes.     

Sequential O-methylation of tricetin by a single gene product in wheat

Flavonoid compounds are ubiquitous in nature. They constitute an important part of the human diet and act as active principles of many medicinal plants. Their O-methylation increases their lipophilicity and hence, their compartmentation and functional diversity. We have isolated and characterized a full-length flavonoid O-methyltransferase cDNA (TaOMT2) from a wheat leaf cDNA library. The recombinant TaOMT2 protein was purified to near homogeneity and tested for its substrate preference against a number of phenolic compounds. Enzyme assays and kinetic analyses indicate that TaOMT2 exhibits a pronounced preference for the flavone, tricetin and gives rise to three methylated enzyme reaction products that were identified by TLC, HPLC and ESI-MS/MS as its mono-, di- and trimethyl ether derivatives. The sequential order of tricetin methylation by TaOMT2 is envisaged to proceed via its 3′-mono- → 3′,5′-di- → 3′,4′,5′-trimethyl ether derivatives. To our knowledge, this is the first report of a gene product that catalyzes three sequential O-methylations of a flavonoid substrate.     

Expression of DNA Damage Response Molecules PARP1, γH2AX,BRCA1, and BRCA2 Predicts Poor Survival of Breast Carcinoma Patients

BACKGROUND: Poly(ADP-ribose) polymerase 1 (PARP1), γH2AX, BRCA1, and BRCA2 are conventional molecular indicators of DNA damage in cells and are often overexpressed in various cancers. In this study, we aimed, using immunohistochemical detection, whether the co-expression of PARP1, γH2AX, BRCA1, and BRCA2 in breast carcinoma (BCA) tissue can provide more reliable prediction of survival of BCA patients. MATERIALS AND METHODS: We investigated immunohistochemical expression and prognostic significance of the expression of PARP1, γH2AX, BRCA1, and BRCA2 in 192 cases of BCAs. RESULTS: The expression of these four molecules predicted earlier distant metastatic relapse, shorter overall survival (OS), and relapse-free survival (RFS) by univariate analysis. Multivariate analysis revealed the expression of PARP1, γH2AX, and BRCA2 as independent poor prognostic indicators of OS and RFS. In addition, the combined expressional pattern of BRCA1, BRCA2, PARP1, and γH2AX (CSbbph) was an additional independent prognostic predictor for OS (P < .001) and RFS (P < .001). The 10-year OS rate was 95% in the CSbbph-low (CSbbph scores 0 and 1) subgroup, but that was only 35% in the CSbbph-high (CSbbph score 4) subgroup. CONCLUSION: This study has demonstrated that the individual and combined expression patterns of PARP1, γH2AX, BRCA1, and BRCA2 could be helpful in determining an accurate prognosis for BCA patients and for the selection of BCA patients who could potentially benefit from anti-PARP1 therapy with a combination of genotoxic chemotherapeutic agents.     

Fatal opioid overdoses during and shortly after hospital admissions in England: A case-crossover study

BackgroundHospital patients who use illicit opioids such as heroin may use drugs during an admission or leave the hospital in order to use drugs. There have been reports of patients found dead from drug poisoning on the hospital premises or shortly after leaving the hospital. This study examines whether hospital admission and discharge are associated with increased risk of opioid-related death.Methods and findingsWe conducted a case-crossover study of opioid-related deaths in England. Our study included 13,609 deaths between January 1, 2010 and December 31, 2019 among individuals aged 18 to 64. For each death, we sampled 5 control days from the period 730 to 28 days before death. We used data from the national Hospital Episode Statistics database to determine the time proximity of deaths and control days to hospital admissions. We estimated the association between hospital admission and opioid-related death using conditional logistic regression, with a reference category of time neither admitted to the hospital nor within 14 days of discharge. A total of 236/13,609 deaths (1.7%) occurred following drug use while admitted to the hospital. The risk during hospital admissions was similar or lower than periods neither admitted to the hospital nor recently discharged, with odds ratios 1.03 (95% CI 0.87 to 1.21; p = 0.75) for the first 14 days of an admission and 0.41 (95% CI 0.30 to 0.56; p < 0.001) for days 15 onwards. 1,088/13,609 deaths (8.0%) occurred in the 14 days after discharge. The risk of opioid-related death increased in this period, with odds ratios of 4.39 (95% CI 3.75 to 5.14; p < 0.001) on days 1 to 2 after discharge and 2.09 (95% CI 1.92 to 2.28; p < 0.001) on days 3 to 14. 11,629/13,609 deaths (85.5%) did not occur close to a hospital admission, and the remaining 656/13,609 deaths (4.8%) occurred in hospital following admission due to drug poisoning. Risk was greater for patients discharged from psychiatric admissions, those who left the hospital against medical advice, and those leaving the hospital after admissions of 7 days or more. The main limitation of the method is that it does not control for time-varying health or drug use within individuals; therefore, hospital admissions coinciding with high-risk periods may in part explain the results.ConclusionsDischarge from the hospital is associated with an acute increase in the risk of opioid-related death, and 1 in 14 opioid-related deaths in England happens in the 2 weeks after the hospital discharge. This supports interventions that prevent early discharge and improve linkage with community drug treatment and harm reduction services.

In a case-crossover study, Dan Lewer and coauthors investigate factors associated with fatal opioid overdoses during and shortly after hospital admissions in England.     

Interaction of the DNA bases and their mononucleotides with pyridine-2-carbaldehyde thiosemicarbazonecopper(II) complexes. Structure of the cytosine derivative

Experimental studies of the binding interactions of [CuL(NO₃)] and [{CuL′(NO₃)}₂] (HL = pyridine-2-carbaldehyde thiosemicarbazone, and HL′ = pyridine-2-carbaldehyde 4N-methylthiosemicarbazone) with adenine, guanine, cytosine, thymine and their mononucleotides (dNMP), 2-deoxyadenosine-5′-monophosphate, (dAMP), 2′-deoxyguanosine-5′-monophosphate, (dGMP), 2′-deoxycytidine-5′-monophpsphate (dCMP), and thymidine-5′-monophosphate (dTMP) have been carried out in aqueous solution at pH 6.0, I = 0.1 M (NaClO₄) and T = 25 °C. The complexation constants of these compounds, calculated by Hildebrand-Benesi plots for the dye binding, D, ([CuL] or [CuL′]) to the nucleobases or nucleotides (P), have shown two linear stretches in adenine, guanine, dAMP and dGMP. The data were analyzed in terms of formation of 1:1 DP and 1:2 DP₂ complexes with increasing purine base or nucleotide content. For cytosine and dCMP only 1:1 complexes have been observed, whereas for thymine and dTMP such complex structures were not observed. The [CuL(Hcyt)](ClO₄) cytosine derivative has been isolated and characterized. The crystal structure consists of perchlorate ions and [CuL(Hcyt)]⁺ monomers attached by hydrogen bond, chelate π−ring and anion-π interactions. The Cu²⁺ ions bind to the NNS chelating moiety of the thiosemicarbazone ligand and the cytosine N13 site (N3, most common notation) yielding a square-planar geometry. A pseudocoordination to the cytosine O12 site (=O2) can also be considered.     

Molecular determinants of substrate specificity in plant 5'-methylthioadenosine nucleosidases

5′-Methylthioadenosine (MTA)/S-adenosylhomocysteine (SAH) nucleosidase (MTAN) is essential for cellular metabolism and development in many bacterial species. While the enzyme is found in plants, plant MTANs appear to select for MTA preferentially, with little or no affinity for SAH. To understand what determines substrate specificity in this enzyme, MTAN homologues from Arabidopsis thaliana (AtMTAN1 and AtMTAN2, which are referred to as AtMTN1 and AtMTN2 in the plant literature) have been characterized kinetically. While both homologues hydrolyze MTA with comparable kinetic parameters, only AtMTAN2 shows activity towards SAH. AtMTAN2 also has higher catalytic activity towards other substrate analogues with longer 5′-substituents. The structures of apo AtMTAN1 and its complexes with the substrate- and transition-state-analogues, 5′-methylthiotubercidin and formycin A, respectively, have been determined at 2.0-1.8 Å resolution. A homology model of AtMTAN2 was generated using the AtMTAN1 structures. Comparison of the AtMTAN1 and AtMTAN2 structures reveals that only three residues in the active site differ between the two enzymes. Our analysis suggests that two of these residues, Leu181/Met168 and Phe148/Leu135 in AtMTAN1/AtMTAN2, likely account for the divergence in specificity of the enzymes. Comparison of the AtMTAN1 and available Escherichia coli MTAN (EcMTAN) structures suggests that a combination of differences in the 5′-alkylthio binding region and reduced conformational flexibility in the AtMTAN1 active site likely contribute to its reduced efficiency in binding substrate analogues with longer 5′-substituents. In addition, in contrast to EcMTAN, the active site of AtMTAN1 remains solvated in its ligand-bound forms. As the apparent pK_a of an amino acid depends on its local environment, the putative catalytic acid Asp225 in AtMTAN1 may not be protonated at physiological pH and this suggests the transition state of AtMTAN1, like human MTA phosphorylase and Streptococcus pneumoniae MTAN, may be different from that found in EcMTAN.     

Effects of terbium chelate structure on dipicolinate ligation and the detection of Bacillus spores

Terbium-sensitized luminescence and its applicability towards the detection of Bacillus spores such as anthrax are of significant interest to research in biodefense and medical diagnostics. Accordingly, we have measured the effects of terbium chelation upon the parameters associated with dipicolinate ligation and spore detection. Namely, the dissociation constants, intrinsic brightness, luminescent lifetimes, and biological stabilities for several Tb(chelate)(dipicolinate)_x complexes were determined using linear, cyclic, and aromatic chelators of differing structure and coordination number. This included the chelator array of NTA, BisTris, EGTA, EDTA, BAPTA, DO2A, DTPA, DO3A, and DOTA (respectively, 2,2′,2″-nitrilotriacetic acid; 2,2-bis(hydroxymethyl)-2,2′,2″-nitrilotriethanol; ethylene glycol-bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid; ethylenediamine-N,N,N′,N′-tetraacetic acid; 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; 1,4,7,10-tetraazacyclododecane-1,7-diacetic acid; diethylenetriamine-N,N,N′,N″,N″-pentaacetic acid; 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid; and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid). Our study has revealed that the thermodynamic and temporal emission stabilities of the Tb(chelate)(dipicolinate)_x complexes are directly related to chelate rigidity and a ligand stoichiometry of x = 1, and that chelators possessing either aromaticity or low coordination numbers are destabilizing to the complexes when in extracts of an extremotolerant Bacillus spore. Together, our results demonstrate that both Tb(EDTA) and Tb(DO2A) are chemically and biochemically stable and thus applicable as respectively low and high-cost luminescent reporters for spore detection, and thereby of significance to institutions with developing biodefense programs.