Two novel dATP analogs for DNA photoaffinity labeling

Two new photoreactive dATP analogs, N⁶-[4-azidobenzoyl–(2-aminoethyl)]-2′-deoxyadenosine-5′-triphosphate (AB-dATP) and N⁶-[4-[3-(trifluoromethyl)-diazirin-3-yl]benzoyl-(2-aminoethyl)]-2′-deoxyadenosine-5′-triphosphate (DB-dATP), were synthesized from 2′-deoxyadenosine-5′-monophosphate in a six step procedure. Synthesis starts with aminoethylation of dAMP and continues with rearrangement of N¹-(2-aminoethyl)-2′-deoxyadenosine-5′-monophosphate to N⁶-(2-aminoethyl)-2′-deoxyadenosine-5′-monophosphate (N⁶-dAMP). Next, N⁶-dAMP is converted into the triphosphate form by first protecting the N-6 primary amino group before coupling the pyrophosphate. After pyrophosphorylation, the material is deprotected to yield N⁶-(2-aminoethyl)-2′-deoxyadenosine-5′-triphosphate (N⁶-dATP). The N-6 amino group is subsequently used to attach either a phenylazide or phenyldiazirine and the photoreactive nucleotide is then enzymatically incorporated into DNA. N⁶-dATP and its photoreactive analogs AB-dATP and DB-dATP were successfully incorporated into DNA using the exonuclease-free Klenow fragment of DNA polymerase I in a primer extension reaction. UV irradiation of the primer extension reaction with AB-dATP or DB-dATP showed specific photocrosslinking of DNA polymerase I to DNA.     

Association of E/E′ and NT-proBNP with Renal Function in Patients with Essential Hypertension

Objectives

To evaluate the association of left ventricular (LV) diastolic function and N-terminal pro-brain natriuretic peptide (NT-proBNP) with renal function in essential hypertension.

Methods

LV diastolic function was estimated by the ratio of early diastolic velocities (E) from transmitral inflow to early diastolic velocities (E′) of tissue Doppler at mitral annulus (septal corner); NT-proBNP was measured in 207 hypertensive patients (mean age 56±14 years). The subjects were classified into 3 groups: E/E′≤10 group (n = 48), 10<E/E′≤15 group (n = 109) and E/E′>15 group (n = 50). The renal function was estimated by glomerular filtration rate (GFR) with ^99mTc-DTPA. GFR from 30 to 59 ml/min/1.73 m² was defined as Stage 3 chronic kidney disease (CKD). GFR was also estimated using the modified MDRD equation. Albuminuria was defined by urinary albumin/creatinine ratio (UACR).

Results

GFR was lower and UACR was higher in E/E′ >15 group than in 10< E/E′ ≤15 group or E/E′ ≤10 group (p<0.0001), GFR was significantly negative and UACR was positive correlated with E/E′ and NT-proBNP (p<0.0001). In multivariate stepwise linear analysis, GFR had significant correlation with age (p = 0.001), gender (p = 0.003), E/E′ (p = 0.03), lgNT-proBNP (p = 0.001) and lgUACR (p = 0.01), while eGFR had no significant correlation with E/E′ or lgNT-proBNP. Multivariate logistic regression analysis, adjusted for potential confounding factors, showed that participants in E/E′>15 group were more likely to have Stage 3 CKD compared with those in E/E′≤10 group with an adjusted odds ratio of 8.31 (p = 0.0036).

Conclusions

LV diastolic function, assessed with E/E′ and NT-proBNP is associated with renal function in essential hypertension.     

Stilbene Synthase and Chalcone Synthase : Two Different Constitutive Enzymes in Cultured Cells of Picea excelsa

Cultured cells of Picea excelsa capable of forming stilbenes and flavanoids have been established. Unlike needles of intact plants containing piceatannol (3,3′,4′,5-tetrahydroxystilbene) and stilbene glycosides the cultured cells converted phenylalanine and p-coumaric acid primarily into resveratrol monomethyl ether (3,4′-dihydroxy-5-methoxystilbene) and naringenin. Partially purified enzyme preparations were assayed for chalcone synthase as well as for stilbene synthase activity converting malonyl-CoA plus p-coumaroyl-CoA into 3,4′,5-trihydroxystilbene (resveratrol).

Although stilbene synthase and chalcone synthase use the same substrates and exhibit similar molecular properties, i.e. molecular weight and subunit molecular weight, they are two different proteins. This difference was demonstrated by gel electrophoresis and by means of monospecific antibodies.

     

Sequence-specific recognition of DNA minor groove by an NIR-fluorescence switch-on probe and its potential applications

In molecular biology, understanding the functional and structural aspects of DNA requires sequence-specific DNA binding probes. Especially, sequence-specific fluorescence probes offer the advantage of real-time monitoring of the conformational and structural reorganization of DNA in living cells. Herein, we designed a new class of D2A (one-donor-two-acceptor) near-infrared (NIR) fluorescence switch-on probe named quinone cyanine–dithiazole (QCy–DT) based on the distinctive internal charge transfer (ICT) process for minor groove recognition of AT-rich DNA. Interestingly, QCy–DT exhibited strong NIR-fluorescence enhancement in the presence of AT-rich DNA compared to GC-rich and single-stranded DNAs. We show sequence-specific minor groove recognition of QCy–DT for DNA containing 5′-AATT-3′ sequence over other variable (A/T)4 sequences and local nucleobase variation study around the 5′-X(AATT)Y-3′ recognition sequence revealed that X = A and Y = T are the most preferable nucleobases. The live cell imaging studies confirmed mammalian cell permeability, low-toxicity and selective staining capacity of nuclear DNA without requiring RNase treatment. Further, Plasmodium falciparum with an AT-rich genome showed specific uptake with a reasonably low IC₅₀ value (<4 µM). The ease of synthesis, large Stokes shift, sequence-specific DNA minor groove recognition with switch-on NIR-fluorescence, photostability and parasite staining with low IC₅₀ make QCy–DT a potential and commercially viable DNA probe.     

Affinity chromatography of bovine trypsin. A rapid separation of bovine α- and β-trypsin

Affinity adsorbents for bovine trypsin were prepared by covalently coupling p-(p′-amino-phenoxypropoxy)benzamidine to cellulose and to agarose. Trypsin binds to both adsorbents at pH6–8 and is released at low pH values or in the presence of n-butylamine hydrochloride. Pure β-trypsin may be eluted from crude trypsin bound at pH8.0 to the cellulose adsorbent by stepwise elution with an acetate buffer, pH5.0. Both α- and β-trypsin may be isolated by chromatography of crude trypsin on the agarose derivative in an acetate buffer, pH4.0. These two methods for purifying the trypsin are specific to the particular adsorbents. They are rapid and convenient in use. Both methods leave a mixture of the two enzymes bound to the adsorbent and release occurs only at low pH values. The effects of pH, composition and ionic strength of buffer and other variables on both purification methods are described. Affinity adsorbents of soya-bean trypsin inhibitor and of N-α-(N′-methyl-N′-sulphanilyl) sulphanilylagmatine bound to agarose were prepared, but were found to be of limited usefulness in the purification of trypsin.     

Poly(U) RNA-templated synthesis of AppA

Simple nucleotide templating activities are of interest as potential primordial reactions. Here we describe the acceleration of 5′-5′ AppA synthesis by 3′-5′ poly(U) under normal solution conditions. This reaction is apparently templated via complementary U:A base-pairing, despite the involvement of two different RNA backbones, because poly(U), unlike other polymers, significantly stimulates AppA synthesis. These interactions occur in moderate (K⁺) and (Mg²⁺) and are temperature sensitive, being more efficient at 10°C than at 4°C, but absent at 20°C. The reaction is only slightly pH sensitive, despite potentially relevant substrate pK_a’s. Kinetic data explicitly support production of AppA by interaction of stacked 2MeImpA and pA nucleotides paired with a single molecule of U template. At a lower rate, AppA can also be produced by a chemical reaction between 2MeImpA and pA, without participation of poly(U). Molecular modeling suggests that 5′-5′ joining between stacked or concurrently paired A''s can occur without major departures from normal U-A helical coordinates. So, coenzyme-like 5′-5′ purine dinucleotides might be readily synthesized from 3′-5′ RNAs with complementary sequences.     

The Putative Nucleic Acid Helicase Sen1p Is Required for Formation and Stability of Termini and for Maximal Rates of Synthesis and Levels of Accumulation of Small Nucleolar RNAs in Saccharomyces cerevisiae

Sen1p from Saccharomyces cerevisiae is a nucleic acid helicase related to DEAD box RNA helicases and type I DNA helicases. The temperature-sensitive sen1-1 mutation located in the helicase motif alters the accumulation of pre-tRNAs, pre-rRNAs, and some small nuclear RNAs. In this report, we show that cells carrying sen1-1 exhibit altered accumulation of several small nucleolar RNAs (snoRNAs) immediately upon temperature shift. Using Northern blotting, RNase H cleavage, primer extension, and base compositional analysis, we detected three forms of the snoRNA snR13 in wild-type cells: an abundant TMG-capped 124-nucleotide (nt) mature form (snR13F) and two less abundant RNAs, including a heterogeneous population of ~1,400-nt 3′-extended forms (snR13R) and a 108-nt 5′-truncated form (snR13T) that is missing 16 nt at the 5′ end. A subpopulation of snR13R contains the same 5′ truncation. Newly synthesized snR13R RNA accumulates with time at the expense of snR13F following temperature shift of sen1-1 cells, suggesting a possible precursor-product relationship. snR13R and snR13T both increase in abundance at the restrictive temperature, indicating that Sen1p stabilizes the 5′ end and promotes maturation of the 3′ end. snR13F contains canonical C and D boxes common to many snoRNAs. The 5′ end of snR13T and the 3′ end of snR13F reside within C₂U₄ sequences that immediately flank the C and D boxes. A mutation in the 5′ C₂U₄ repeat causes underaccumulation of snR13F, whereas mutations in the 3′ C₂U₄ repeat cause the accumulation of two novel RNAs that migrate in the 500-nt range. At the restrictive temperature, double mutants carrying sen1-1 and mutations in the 3′ C₂U₄ repeat show reduced accumulation of the novel RNAs and increased accumulation of snR13R RNA, indicating that Sen1p and the 3′ C₂U₄ sequence act in a common pathway to facilitate 3′ end formation. Based on these findings, we propose that Sen1p and the C₂U₄ repeats that flank the C and D boxes promote maturation of the 3′ terminus and stability of the 5′ terminus and are required for maximal rates of synthesis and levels of accumulation of mature snR13F.     

Estrogen-induced upregulation and 3′-UTR shortening of CDC6

3′-Untranslated region (UTR) shortening of mRNAs via alternative polyadenylation (APA) has important ramifications for gene expression. By using proximal APA sites and switching to shorter 3′-UTRs, proliferating cells avoid miRNA-mediated repression. Such APA and 3′-UTR shortening events may explain the basis of some of the proto-oncogene activation cases observed in cancer cells. In this study, we investigated whether 17 β-estradiol (E2), a potent proliferation signal, induces APA and 3′-UTR shortening to activate proto-oncogenes in estrogen receptor positive (ER+) breast cancers. Our initial probe based screen of independent expression arrays suggested upregulation and 3′-UTR shortening of an essential regulator of DNA replication, CDC6 (cell division cycle 6), upon E2 treatment. We further confirmed the E2- and ER-dependent upregulation and 3′UTR shortening of CDC6, which lead to increased CDC6 protein levels and higher BrdU incorporation. Consequently, miRNA binding predictions and dual luciferase assays suggested that 3′-UTR shortening of CDC6 was a mechanism to avoid 3′-UTR-dependent negative regulations. Hence, we demonstrated CDC6 APA induction by the proliferative effect of E2 in ER+ cells and provided new insights into the complex regulation of APA. E2-induced APA is likely to be an important but previously overlooked mechanism of E2-responsive gene expression.     

Influence of Serotonin Transporter Gene Polymorphisms and Adverse Life Events on Depressive Symptoms in the Elderly: A Population-Based Study

BackgroundDepression is common in the elderly. The role of genetic and environmental factors in modulating depressive symptoms is not clear.MethodsWe evaluated the influence of serotonin transporter gene polymorphisms and recent adverse life events on depressive symptoms in an elderly Italian population. We used data from “InveCe.Ab”, a population-based study of 1321 subjects aged 70–74 years. We used the 15-item Geriatric Depression Scale (GDS) to assess depressive symptoms–a GDS score ≥5 points (GDS≥5) indicated the presence of clinically relevant symptoms–and performed 5-HTTLPR and rs25531 genotyping to obtain the triallelic polymorphism of the serotonin transporter. We used the Geriatric Adverse Life Events Scale to measure adverse life events, and logistic regression models to evaluate the role of genotype and recent adverse life events in depressive symptoms, controlling for potential confounders and independent predictors.ResultsTwo hundred subjects (15.76%) had a GDS≥5. The 5-HTTLPR triallelic polymorphism was significantly associated with GDS≥5. Only S′S′ carriers showed an increased risk of depressive symptoms (OR_adj = 1.81, p = .022); one extra adverse life event increased this risk by 14% (p = .061) independently of genotype. Other factors significantly related to GDS≥5 were: female gender (OR_adj = 2.49, p < .001), age (OR_adj = 1.19, p = .007), a history of depression (OR_adj = 4.73, p < .001), and comorbidity (OR_adj = 1.23, p = .001). One extra adverse life event increased the risk of depressive symptoms by 57% (p = .005) only in the L′L′ carriers, while antidepressant intake was directly related to GDS≥5 in the L′S′ carriers (OR_adj = 2.46, p = .036) and borderline significant in the S′S′ carriers (OR_adj = 2.41, p = .081).DiscussionThe S′S′ genotype and recent exposure to adverse life events were independently associated with depressive symptoms. The S′S′ genotype, compared with the environment, exerted a predominant effect on depressive symptoms, suggesting that it reduces the efficacy of antidepressant therapy. We conclude that genetics may be an important risk factor for depressive symptoms in late adulthood.     

Characterization of the 2',3' cyclic phosphodiesterase activities of Clostridium thermocellum polynucleotide kinase-phosphatase and bacteriophage lambda phosphatase

Clostridium thermocellum polynucleotide kinase-phosphatase (CthPnkp) catalyzes 5′ and 3′ end-healing reactions that prepare broken RNA termini for sealing by RNA ligase. The central phosphatase domain of CthPnkp belongs to the dinuclear metallophosphoesterase superfamily exemplified by bacteriophage λ phosphatase (λ-Pase). CthPnkp is a Ni²⁺/Mn²⁺-dependent phosphodiesterase-monoesterase, active on nucleotide and non-nucleotide substrates, that can be transformed toward narrower metal and substrate specificities via mutations of the active site. Here we characterize the Mn²⁺-dependent 2′,3′ cyclic nucleotide phosphodiesterase activity of CthPnkp, the reaction most relevant to RNA repair pathways. We find that CthPnkp prefers a 2′,3′ cyclic phosphate to a 3′,5′ cyclic phosphate. A single H189D mutation imposes strict specificity for a 2′,3′ cyclic phosphate, which is cleaved to form a single 2′-NMP product. Analysis of the cyclic phosphodiesterase activities of mutated CthPnkp enzymes illuminates the active site and the structural features that affect substrate affinity and k_cat. We also characterize a previously unrecognized phosphodiesterase activity of λ-Pase, which catalyzes hydrolysis of bis-p-nitrophenyl phosphate. λ-Pase also has cyclic phosphodiesterase activity with nucleoside 2′,3′ cyclic phosphates, which it hydrolyzes to yield a mixture of 2′-NMP and 3′-NMP products. We discuss our results in light of available structural and functional data for other phosphodiesterase members of the binuclear metallophosphoesterase family and draw inferences about how differences in active site composition influence catalytic repertoire.     

Purification of a trifunctional enzyme, catalysing three steps of the histidine pathway, from Neurospora crassa

1. A procedure is described for the purification of an enzyme from Neurospora crassa that has three catalytic functions. These are 1-N-(5′-phosphoribosyl)-ATP pyrophosphohydrolase, 1-N-(5′-phosphoribosyl)-AMP cyclohydrolase and histidinol dehydrogenase (l-histidinol–NAD oxidoreductase, EC 1.1.1.23), and are responsible for the catalysis of reactions 2, 3 and 10 in the histidine pathway. The ratio of these three catalytic activities remains approximately the same throughout the purification procedure. Evidence is presented that the purified preparations contain a single protein exhibiting association–dissociation equilibria.     

Strand directionality affects cation binding and movement within tetramolecular G-quadruplexes

Nuclear magnetic resonance study of G-quadruplex structures formed by d(TG₃T) and its modified analogs containing a 5′-5′ or 3′-3′ inversion of polarity sites, namely d(3′TG5′-5′G₂T3′), d(3′T5′-5′G₃T3′) and d(5′TG3′-3′G₂T5’) demonstrates formation of G-quadruplex structures with tetrameric topology and distinct cation-binding preferences. All oligonucleotides are able to form quadruplex structures with two binding sites, although the modified oligonucleotides also form, in variable amounts, quadruplex structures with only one bound cation. The inter-quartet cavities at the inversion of polarity sites bind ammonium ions less tightly than a naturally occurring 5′-3′ backbone. Exchange of ¹⁵ ions between G-quadruplex and bulk solution is faster at the 3′-end in comparison to the 5′-end. In addition to strand directionality, cation movement is influenced by formation of an all-syn G-quartet. Formation of such quartet has been observed also for the parent d(TG₃T) that besides the canonical quadruplex with only all-anti G-quartets, forms a tetramolecular parallel quadruplex containing one all-syn G-quartet, never observed before in unmodified quadruplex structures.     

Triplex-induced recombination and repair in the pyrimidine motif

Triplex-forming oligonucleotides (TFOs) bind DNA in a sequence-specific manner at polypurine/polypyrimidine sites and mediate targeted genome modification. Triplexes are formed by either pyrimidine TFOs, which bind parallel to the purine strand of the duplex (pyrimidine, parallel motif), or purine TFOs, which bind in an anti-parallel orientation (purine, anti-parallel motif). Both purine and pyrimidine TFOs, when linked to psoralen, have been shown to direct psoralen adduct formation in cells, leading to mutagenesis or recombination. However, only purine TFOs have been shown to mediate genome modification without the need for a targeted DNA-adduct. In this work, we report the ability of a series of pyrimidine TFOs, with selected chemical modifications, to induce repair and recombination in two distinct episomal targets in mammalian cells in the absence of any DNA-reactive conjugate. We find that TFOs containing N3′→P5′ phosphoramidate (amidate), 5-(1-propynyl)-2′-deoxyuridine (pdU), 2′-O-methyl-ribose (2′-O-Me), 2′-O-(2-aminoethyl)-ribose, or 2′-O, 4′-C-methylene bridged or locked nucleic acid (LNA)-modified nucleotides show substantially increased formation of non-covalent triplexes under physiological conditions compared with unmodified DNA TFOs. However, of these modified TFOs, only the amidate and pdU-modified TFOs mediate induced recombination in cells and stimulate repair in cell extracts, at levels comparable to those seen with purine TFOs in similar assays. These results show that amidate and pdU-modified TFOs can be used as reagents to stimulate site-specific gene targeting without the need for conjugation to DNA-reactive molecules. By demonstrating the potential for induced repair and recombination with appropriately modified pyrimidine TFOs, this work expands the options available for triplex-mediated gene targeting.     

Metabolism of Cytokinin : DEPHOSPHORYLATION OF CYTOKININ RIBONUCLEOTIDE BY 5'-NUCLEOTIDASES FROM WHEAT GERM CYTOSOL

Two forms (F-I and F-II) of 5′-nucleotidases (5′-ribonucleotide phosphohydrolase, EC 3.1.3.5) which catalyze the dephosphorylation of N⁶-(Δ²-isopentenyl)adenosine 5′-monophosphate and AMP to form the corresponding nucleosides were partially purified from the cytosol of wheat (Triticum aestivum) germ. Both the F-I (molecular weight, 57,000) and F-II (molecular weight, 110,000) 5′-nucleotidases dephosphorylate the ribonucleotides at an optimum pH of 7. The K_m values for the cytokinin nucleotide are 3.5 micromolar (F-I enzyme) and 12.8 micromolar (F-II enzyme) in 100 millimolar Tris-maleate buffer (pH 7) at 37 C. The F-I enzyme is less rapidly inactivated by heating than is the F-II enzyme. Both nucleotidases hydrolyze purine ribonucleoside 5′-phosphates, AMP being the preferred substrate. N⁶-(Δ²-isopentenyl)Adenosine 5′-monophosphate is hydrolyzed at a rate 72 and 86% that of AMP by the F-I and F-II nucleotides, respectively. Phenylphosphate and 3′-AMP are not substrates for the enzymes. It is proposed that dephosphorylation of cytokinin nucleotide by cytosol 5′-nucleotidases may play an important role in regulating levels of “active cytokinin” in plant cells.     

MicroRNA-18a Attenuates DNA Damage Repair through Suppressing the Expression of Ataxia Telangiectasia Mutated in Colorectal Cancer

Background

miR-18a is one of the most up-regulated miRNAs in colorectal cancers (CRC) based on miRNA profiling. In this study, we examined the functional significance of miR-18a in CRC.

Methods

Expression of miR-18a was investigated in 45 CRC patients. Potential target genes of miR-18a were predicted by in silico search and confirmed by luciferase activity assay and Western blot. DNA damage was measured by comet assay. Gene function was measured by cell viability, colony formation and apoptosis assays.

Results

The up-regulation of miR-18a was validated and confirmed in 45 primary CRC tumors compared with adjacent normal tissues (p<0.0001). Through in silico search, the 3′UTR of Ataxia telangiectasia mutated (ATM) contains a conserved miR-18a binding site. Expression of ATM was down-regulated in CRC tumors (p<0.0001) and inversely correlated with miR-18a expression (r = -0.4562, p<0.01). Over-expression of miR-18a in colon cancer cells significantly reduced the luciferase activity of the construct with wild-type ATM 3′UTR but not that with mutant ATM 3′UTR, inferring a direct interaction of miR-18a with ATM 3′UTR. This was further confirmed by the down-regulation of ATM protein by miR-18a. As ATM is a key enzyme in DNA damage repair, we evaluated the effect of miR-18a on DNA double-strand breaks. Ectopic expression of miR-18a significantly inhibited the repair of DNA damage induced by etoposide (p<0.001), leading to accumulation of DNA damage, increase in cell apoptosis and poor clonogenic survival.

Conclusion

miR-18a attenuates cellular repair of DNA double-strand breaks by directly suppressing ATM, a key enzyme in DNA damage repair.     

Hydroxylation of the Herbicide Isoproturon by Fungi Isolated from Agricultural Soil

Several asco-, basidio-, and zygomycetes isolated from an agricultural field were shown to be able to hydroxylate the phenylurea herbicide isoproturon [N-(4-isopropylphenyl)-N′,N′-dimethylurea] to N-(4-(2-hydroxy-1-methylethyl)phenyl)-N′,N′-dimethylurea and N-(4-(1-hydroxy-1-methylethyl)phenyl)-N′,N′-dimethylurea. Bacterial metabolism of isoproturon has previously been shown to proceed by an initial demethylation to N-(4-isopropylphenyl)-N′-methylurea. In soils, however, hydroxylated metabolites have also been detected. In this study we identified fungi as organisms that potentially play a major role in the formation of these hydroxylated metabolites in soils treated with isoproturon. Isolates of Mortierella sp. strain Gr4, Phoma cf. eupyrena Gr61, and Alternaria sp. strain Gr174 hydroxylated isoproturon at the first position of the isopropyl side chain, yielding N-(4-(2-hydroxy-1-methylethyl)phenyl)-N′,N′-dimethylurea, while Mucor sp. strain Gr22 hydroxylated the molecule at the second position, yielding N-(4-(1-hydroxy-1-methylethyl)phenyl)-N′,N′-dimethylurea. Hydroxylation was the dominant mode of isoproturon transformation in these fungi, although some cultures also produced traces of the N-demethylated metabolite N-(4-isopropylphenyl)-N′-methylurea. A basidiomycete isolate produced a mixture of the two hydroxylated and N-demethylated metabolites at low concentrations. Clonostachys sp. strain Gr141 and putative Tetracladium sp. strain Gr57 did not hydroxylate isoproturon but N demethylated the compound to a minor extent. Mortierella sp. strain Gr4 also produced N-(4-(2-hydroxy-1-methylethyl)phenyl)-N′-methylurea, which is the product resulting from combined N demethylation and hydroxylation.