Studies on the substrate specificity of the T 4 excision repair endonuclease

Previous studies have shown that the v gene of bacteriophage T4 codes for an endonuclease that specifically attacks pyrimidine dimer sites in UV-irradiated DNA. The present studies have examined the role of this endonuclease in the repair of DNA damaged by nitrogen mustard, N-methyl-N′-nitro-N-nitrosoguanidine (NTG), mitomycin C and 4-nitroquinoline-N-oxide. The observation by Harm that the v gene product of phage T4 facilitates repair of UV damage to the host DNA of excision-repair defective strains enabled us to test whether it does the same with other cellular DNA lesions. It was shown that infection of UV-irradiated E. coliB_s−1 with UV-inactivated phage T4v+ resulted in rescue of a certain fraction of the host cells. However no v gene mediated repair E. coli B_s−1 was observed following treatment with the chemical agents mentioned. Furthermore, though phage T4v₁ is more sensitive to UV-irradiation than phage T4, there was no observed difference in the sensitivity of these phages to nitrogen mustard or NTG. On the basis of these observations it was concluded that the v gene coded endonuclease of T4 is specific for the excision repair of pyrimidine dimers and does not participate in the repair of chemically damaged DNA. In vitro enzymatic degradation of DNA alkylated with nitrogen mustard was observed, but it is probable that this degradation is not part of a repair reaction in vivo.     

Structural Insights into Substrate Specificity in Variants of N-Acetylneuraminic Acid Lyase Produced by Directed Evolution

The substrate specificity of Escherichia coli N-acetylneuraminic acid lyase was previously switched from the natural condensation of pyruvate with N-acetylmannosamine, yielding N-acetylneuraminic acid, to the aldol condensation generating N-alkylcarboxamide analogues of N-acetylneuraminic acid. This was achieved by a single mutation of Glu192 to Asn. In order to analyze the structural changes involved and to more fully understand the basis of this switch in specificity, we have isolated all 20 variants of the enzyme at position 192 and determined the activities with a range of substrates. We have also determined five high-resolution crystal structures: the structures of wild-type E. coli N-acetylneuraminic acid lyase in the presence and in the absence of pyruvate, the structures of the E192N variant in the presence and in the absence of pyruvate, and the structure of the E192N variant in the presence of pyruvate and a competitive inhibitor (2R,3R)-2,3,4-trihydroxy-N,N-dipropylbutanamide. All structures were solved in space group P2₁ at resolutions ranging from 1.65 Å to 2.2 Å. A comparison of these structures, in combination with the specificity profiles of the variants, reveals subtle differences that explain the details of the specificity changes. This work demonstrates the subtleties of enzyme-substrate interactions and the importance of determining the structures of enzymes produced by directed evolution, where the specificity determinants may change from one substrate to another.     

New N-acyl-d-glucosamine 2-epimerases from cyanobacteria with high activity in the absence of ATP and low inhibition by pyruvate

N-Acetylneuraminic acid, an important component of glycoconjugates with various biological functions, can be produced from N-acetyl-d-glucosamine (GlcNAc) and pyruvate using a one-pot, two-enzyme system consisting of N-acyl-d-glucosamine 2-epimerase (AGE) and N-acetylneuraminate lyase (NAL). In this system, the epimerase catalyzes the conversion of GlcNAc into N-acetyl-d-mannosamine (ManNAc). However, all currently known AGEs have one or more disadvantages, such as a low specific activity, substantial inhibition by pyruvate and strong dependence on allosteric activation by ATP. Therefore, four novel AGEs from the cyanobacteria Acaryochloris marina MBIC 11017, Anabaena variabilis ATCC 29413, Nostoc sp. PCC 7120, and Nostoc punctiforme PCC 73102 were characterized. Among these enzymes, the AGE from the Anabaena strain showed the most beneficial characteristics. It had a high specific activity of 117 ± 2 U mg⁻¹ at 37 °C (pH 7.5) and an up to 10-fold higher inhibition constant for pyruvate as compared to other AGEs indicating a much weaker inhibitory effect. The investigation of the influence of ATP revealed that the nucleotide has a more pronounced effect on the K_m for the substrate than on the enzyme activity. At high substrate concentrations (≥200 mM) and without ATP, the enzyme reached up to 32% of the activity measured with ATP in excess.     

Synthesis and characterization of chitosan-homocysteine thiolactone as a mucoadhesive polymer

Two mucoadhesive thiolated polymers were synthesized by the covalent attachment of homocysteine thiolactone (HT) to chitosan and N,N,N-trimethyl-chitosan (TM-chitosan) at various chitosan:HT ratios. The amount of thiol and disulphide groups immobilized on the chitosan influenced the polymer's mucoadhesion positively and negatively, respectively, with the optimal chitosan:HT (w/w) ratio being found to be 1:0.1. The interaction between mucin and chitosan and its three derivatives was highest for the thiolated chitosan derivatives but was pH dependent. HT-chitosan and TM-HT-chitosan, with the thiol groups of 64.15 and 32.48 μmol/g, respectively, displayed a 3.67- and 6.33-fold stronger mucoadhesive property compared to that of the unmodified chitosan at pH 1.2, but these differences were only ∼1.7-fold at pH 6.4. The swelling properties of TM-HT-chitosan and HT-chitosan were higher than that of chitosan and TM-chitosan, attaining a swelling ratio of up to 240% and 140%, respectively, at pH 1.2 within 2 h.     

2-(1-(Dimethylamino)ethyl)phenylpalladium(II) complexes 5-functionalized with fluorous silyl tails

New fluorous-organometallics based on the chiral ligand α-methyl-N,N-dimethylbenzylamine (TMBA) were prepared by treatment of fluorous silyl bromide reagents with in situ 4-lithiated TMBA to give fluorous N,N-dimethyl(α-methyl-4-trialkylsilylbenzyl)amine ligands 1a-1c that vary in the number of fluorous tails attached to the Si atom. Ligands 1a-1c were successfully cyclo-palladated by treatment with Pd(OAc)₂/LiCl in methanol to furnish the corresponding chloride-bridged dimeric arylpalladium(II) complexes 2a-2c in good yields. The latter derivatives could be converted into monomeric Lewis-base adducts by complexation with pyridine (3a-3c), or triphenylphosphine (4a-4c). The crystal structure of triphenylphosphine complex 4a has been elucidated. To probe their fluorophilicity, the partition coefficient of each of the derivatives in the fluorous biphasic solvent (FBS) system perfluoromethylcyclohexane/n-octane has been determined.     

Interactions between enhancer of rudimentary and Notch and deltex reveal a regulatory function of enhancer of rudimentary in the Notch signaling pathway in Drosophila melanogaster

Enhancer of rudimentary, e(r), encodes a small nuclear protein, ER, that has been implicated in the regulation of pyrimidine metabolism, DNA replication and cell proliferation. In Drosophila melanogaster, a new recessive Notch allele, N^nd-p, was isolated as a lethal in combination with an e(r) allele, e(r)^p2. Both mutants are viable as single mutants. N^nd-p is caused by a P-element insertion in the 5′ UTR, 378-bp upstream of the start of translation. Together the molecular and genetic data argue that N^nd-p is a hypomorphic allele of N. The three viable notchoid alleles, N^nd-p, N^nd-1 and N^nd-3, are lethal in combination with e(r)⁻ alleles. Our present hypothesis is that e(r) is a positive regulator of the Notch signaling pathway and that the lethality of the N e(r) double mutants is caused by a reduction in the expression of the pathway. This is supported by the rescue of the lethality by a mutation in Hairless, a negative regulator of N, and by the synthetic lethality of dx e(r) double mutants. Further support for the hypothesis is a reduction in E(spl) expression in an e(r)⁻ mutant. Immunostaining localizes ER to the nucleus, suggesting a nuclear function for ER. A role in the Notch signaling pathway, suggests that e(r) may be expressed in the nervous system. This turns out to be the case, as immunostaining of ER shows that ER is localized to the developing CNS.     

Succinimidyl oleate, established inhibitor of CD36/FAT translocase inhibits complex III of mitochondrial respiratory chain

The functional role of CD36 protein detected in mitochondrial fractions in long chain fatty acid (LCFA) oxidation is unclear due to conflicting results obtained in Cd36 knockout mice and experiments using sulfo-N-succinimidyl oleate (SSO) for inhibition of CD36 mediated LCFA transport. We investigated effect of SSO on mitochondrial respiration and found that SSO substantially inhibits not only LCFA oxidation, but also oxidation of flavoprotein- and NADH-dependent substrates and generation of mitochondrial membrane potential. Experiments in rat liver, heart and kidney mitochondria demonstrated a direct effect on mitochondrial respiratory chain with the most pronounced inhibition of the complex III (IC₅₀ 4 μM SSO). The results presented here show that SSO is a potent and irreversible inhibitor of mitochondrial respiratory chain.     

N-Glycosylation pattern of recombinant human CD82 (KAI1), a tumor-associated membrane protein

The membrane glycoprotein CD82 (KAI1) has attracted increasing attention as a suppressor of cell migration, related tumor invasion, as well as metastasis. The glycosylation of CD82 has been shown to be involved in a correlative cell adhesion and motility. However, the N-glycosylation pattern of CD82 has not been described yet. In the current study, a detailed characterization of the recombinant human CD82 N-linked glycosylation pattern was conducted by employing an integrative proteomic and glycomic approach, including glycosidase and protease digestions, glycan permethylation, MS analyses, site-directed mutagenesis, and lectin blots. The results reveal three N-glycosylation sites, and further demonstrate a putative glycosylation site at Asn₁₅₇ for the first time. A highly heterogeneous pattern of N-linked glycans is described, which express distinct carbohydrate epitopes, such as bisecting N-acetylglucosamine, (α-2,6) N-acetylneuraminic acid, and core fucose. These epitopes are highly associated with various biological functions, including cell adhesion and cancer metastasis, and can possibly influence the anti-cancer inhibition ability of CD82.     

Bacterial Protein N-Glycosylation: New Perspectives and Applications

Protein glycosylation is widespread throughout all three domains of life. Bacterial protein N-glycosylation and its application to engineering recombinant glycoproteins continue to be actively studied. Here, we focus on advances made in the last 2 years, including the characterization of novel bacterial N-glycosylation pathways, examination of pathway enzymes and evolution, biological roles of protein modification in the native host, and exploitation of the N-glycosylation pathways to create novel vaccines and diagnostics.     

X-ray and NMR study of the fate of the Co(1,10-phenanthroline-5,6-diketone)3 ion in aqueous solution: supramolecular motifs in the packing of 1,10-phenanthroline-5,6-diketone and 1,10-phenanthroline-5,6-diol complexes

X-ray structures are presented of three new cobalt complexes prepared from Co(III) and N,N^′-1,10-phenanthroline-5,6-dione. The cis-aqua-chloro-bis(N,N^′-1,10-phenanthroline-5,6-dione)cobalt(II) nitrate trihydrate (3) and the cis-aqua-bromo-bis(N,N^′-1,10-phenanthroline-5,6-dione)cobalt(II) trifluoro-methanesulfonate tetrahydrate (4), crystalize in the same space group with a similar arrangement of the complex ions. However, on the molecular scale there are important differences. The cobalt complex in 3 has a typical high-spin geometry whereas in 4 the cobalt complex displays a Jahn-Teller distortion characteristic for low-spin compounds. The third structure is di(N,N^′-1,10-phenanthroline-5,6-diol)(N,N^′-1,10-phenanthroline-5,6-dione)cobalt(III) bromide hexahydrate (5). NMR studies of the hydration of the Co(III)(1,10-phenanthroline-5,6-dione)₃ ³⁺ ion in water and DMSO are also presented. The various possible transformations of the N,N^′-1,10-phenanthroline-5,6-dione ligand are discussed.     

Production of N-acyl Homoserine Lactones and Virulence Factors of Waterborne Aeromonas hydrophila

Aeromonads are inhabitants of aquatic ecosystems and are described as being involved in intestinal disturbances and other infections. The purpose of this study was to investigate the production of N-acyl-homoserine lactone (AHL) signal molecules and some virulence factors, including hemolysins, proteases, extracellular nucleases production and cytotoxicity by waterborne Aeromonas hydrophila. A total of 24 strains isolated from fresh-water or diseased fish were used in the study. The majority A.hydrophila strains produce two AHL molecules (21/24), one is N-butanoyl homoserine lactone (BHL), and the other is N-hexanoyl homoserine lactone (HHL) according to thin-layer chromatography analysis. Among the virulence factors tested, more than 83 % of the isolates produced β haemolysin when inoculated on sheep blood agar, only 50 % of the isolates displayed DNase activity, 75 % of the isolates shown proteolytic activity on skimmed milk plate, and cytotoxic activity was detected in 20 of 24 of the isolates. The strains producing AHLs possessed one or more virulence factors. In conclusion, the production of quorum sensing signal molecules is common among the strains that we examined, and there seems to some relationships between quorum sensing signal production and virulence factors in A. hydrophila.     

Identifying abnormalities in symbiotic development between Trifolium spp. and Rhizobium leguminosarum bv. trifolii leading to sub-optimal and ineffective nodule phenotypes

Background and Aims

Legumes overcome nitrogen limitations by entering into a mutualistic symbiosis with N₂-fixing bacteria (rhizobia). Fully compatible associations (effective) between Trifolium spp. and Rhizobium leguminosarum bv. trifolii result from successful recognition of symbiotic partners in the rhizosphere, root hair infection and the formation of nodules where N₂-fixing bacteroids reside. Poorly compatible associations can result in root nodule formation with minimal (sub-optimal) or no (ineffective) N₂-fixation. Despite the abundance and persistence of strains in agricultural soils which are poorly compatible with the commercially grown clover species, little is known of how and why they fail symbiotically. The aims of this research were to determine the morphological aberrations occurring in sub-optimal and ineffective clover nodules and to determine whether reduced bacteroid numbers or reduced N₂-fixing activity is the main cause for the Sub-optimal phenotype.

Methods

Symbiotic effectiveness of four Trifolium hosts with each of four R. leguminosarum bv. trifolii strains was assessed by analysis of plant yields and nitrogen content; nodule yields, abundance, morphology and internal structure; and bacteroid cytology, quantity and activity.

Key Results

Effective nodules (Nodule Function 83–100 %) contained four developmental zones and N₂-fixing bacteroids. In contrast, Sub-optimal nodules of the same age (Nodule Function 24–57 %) carried prematurely senescing bacteroids and a small bacteroid pool resulting in reduced shoot N. Ineffective-differentiated nodules carried bacteroids aborted at stage 2 or 3 in differentiation. In contrast, bacteroids were not observed in Ineffective-vegetative nodules despite the presence of bacteria within infection threads.

Conclusions

Three major responses to N₂-fixation incompatibility between Trifolium spp. and R. l. trifolii strains were found: failed bacterial endocytosis from infection threads into plant cortical cells, bacteroid differentiation aborted prematurely, and a reduced pool of functional bacteroids which underwent premature senescence. We discuss possible underlying genetic causes of these developmental abnormalities and consider impacts on N₂-fixation of clovers.     

Different coordination modes of 5,5-diethylbarbiturate in the copper(II) complexes with some aliphatic amines: Synthesis, spectroscopic, thermal and structural studies

Three new copper(II) complexes of 5,5-diethlybarbiturate (barb), [Cu(barb)₂(dmen)]·0.5H₂O (dmen = N,N-dimethylethylenediamine) 1, [Cu(barb)₂(bapa)] (bapa = bis(3-aminopropyl)amine) 2, and [Cu(barb)(apen)](barb)·2H₂O (apen = N,N′-bis(3-aminopropyl)ethylenediamine) 3, have been synthesized and characterized by chemical, spectroscopic and thermal methods. Single crystal X-ray diffraction studies revealed that all complexes are mononuclear. The copper(II) ion exhibits a square-pyramidal coordination geometry in 1 and 3, but a trigonal-bipyramidal geometry in 2. The barb ligand shows different coordination modes. 1 presents the unequal coordination of the barb ligands: one is monodentate (N) and the other one is bidentate (N, O). In 2, both barb ligands are N-coordinated, whereas in 3, one barb ligand is N-coordinated, while the second barb ligand behaves as a counter-ion. The dmen, bapa and apen ligands act as bi-, tri- and tetradentate ligands, respectively. All complexes display a hydrogen-bonded network structure. The IR spectroscopic analysis shows that the ν(CO) stretching frequencies do not correlate predictably with the coordination mode of the barb ligand in 1. Thermal analysis data for 1-3 are in agreement with the crystal structures.     

One-step isolation of sappanol and brazilin from Caesalpinia sappan and their effects on oxidative stress-induced retinal death

Caesalpinia sappan is a well-distributed plant that is cultivated in Southeast Asia, Africa, and the Americas. C. sappan has been used in Asian folk medicine and its extract has been shown to have pharmacological effects. Two homoisoflavonoids, sappanol and brazilin, were isolated from C. sappan by using centrifugal partition chromatography (CPC), and tested for protective effects against retinal cell death. The isolated homoisoflavonoids produced approximately 20-fold inhibition of N-retinylidene-N-retinyl-ethanolamine (A2E) photooxidation in a dose-dependent manner. Of the 2 compounds, brazilin showed better inhibition (197.93 ± 1.59 μM of IC₅₀). Cell viability tests and PI/Hoechst 33342 double staining method indicated that compared to the negative control, sappanol significantly attenuated H₂O₂-induced retinal death. The compounds significantly blunted the up-regulation of intracellular reactive oxygen species (ROS), and sappanol inhibited lipid peroxidation in a concentration-dependent manner. Thus, both compounds represent potential antioxidant treatments for retinal diseases. [BMB Reports 2015; 48(5): 289-294]     

A structural study of the hydrated and the dimethylsulfoxide, N,N′-dimethylpropyleneurea, and N,N-dimethylthioformamide solvated iron(II) and iron(III) ions in solution and solid state

The structures of the solvated iron(II) and iron(III) ions have been studied in solution and solid state by extended X-ray absorption fine structure (EXAFS) in three oxygen donor solvents, water, dimethylsulfoxide (Me₂SO), N,N′-dimethylpropyleneurea (DMPU), and one sulfur donor solvent, N,N-dimethylthioformamide (DMTF); these solvents have different coordination and solvation properties. In addition, the structure of hexakis(dimethylsulfoxide)iron(III) perchlorate has been determined crystallographically to support the determination of the corresponding solvate in solution. The hydrated, the dimethylsulfoxide and N,N-dimethylthioformamide solvated iron(II) ions show regular octahedral coordination in both solution and solid state with mean Fe-O, Fe-O, and Fe-S bond distances of 2.10, 2.10, and 2.52 Å, respectively, whereas the N,N′-dimethylpropyleneurea iron(II) solvate is five-coordinated, d(Fe-O) = 2.06 Å. The compounds vary in color from light green (hydrate) to dark orange or red (DMPU). The hydrated iron(III) ion in aqueous solution and the dimethylsulfoxide solvated iron(III) ions in solution and solid state show the expected octahedral coordination, the Fe-O bond distances are 2.00 Å for both, whereas the N,N′-dimethylpropyleneurea iron(III) solvate is found to be five-coordinated with a mean Fe-O bond distance of 1.99 Å. The N,N-dimethylthioformamide solvated iron(III) ion in the solid perchlorate salt is tetrahedrally four-coordinated, the mean Fe-S bond distance is 2.20 Å. Iron(III) is reduced with time to iron(II) in N,N-dimethylthioformamide solution. The compounds vary in color from pale yellow (hydrate) to blackish red (DMPU).     

Crystal structure and fluorescence of a europium (III) complex: EuCl3(2,2′-bipyridine N,N dioxide) · 2CH3OH

We report the synthesis and characterization of a seven coordinate europium complex, [EuCl₃(C₁₀H₈N₂O₂) ·  2CH₃OH]. The growing interest in developing efficient light conversion molecular devices (LCMDs) necessitates the need for new fluorescent materials. Ideal physicochemical properties of the materials include ligand absorption, efficient metal to ligand transfer, and strong luminescence with a relatively long decay time. The design of such material requires distinct absorbing (ligand) and emitting (metal ion) components. While Eu³⁺ cation has a non-degenerate emitting level, 2,2′-bipyridine N,N dioxide is a heterocyclic ligand known to exhibit strong luminescence. Additional characterization is also described, including single crystal X-ray diffraction, IR and UV-Vis spectroscopies and elemental analysis.     

Synthesis and transition metal complexes of 3,3-bis(1-vinylimidazol-2-yl)propionic acid, a new N,N,O ligand suitable for copolymerisation

The new N,N,O heteroscorpionate ligand 3,3-bis(1-vinylimidazol-2-yl)propionic acid (Hbvip) (5) was synthesised in five steps starting from 1-vinylimidazole. This ligand is closely related to 3,3-bis(1-methylimidazol-2-yl)propionic acid (Hbmip), but contains two vinyl linker groups which can be used for radical-induced polymerisation reactions. The κ³-N,N,O coordination behaviour of 5 was proven by the synthesis of the tricarbonyl complexes [Re(bvip)(CO)₃] (6), [Mn(bvip)(CO)₃] (7) and [Cu(bvip)₂] (8). To obtain good yields of 6, it was synthesised in water instead of THF. The ligand as well as all three complexes were characterised by X-ray crystallography. Copolymerisation of 5 with pure methyl methacrylate (MMA) or a combination of MMA and ethylene glycol dimethacrylate (EGDMA) led to the solid phases P1 and P2. Polymer-bound rhenium and manganese tricarbonyl complexes could be obtained by the reaction of deprotonated P1 with [MBr(CO)₅] (M = Re, Mn) and also by copolymerisation of 6 and 7 with MMA. In both cases, the facial tripodal binding behaviour was evidenced by IR spectra of the polymers. Furthermore, the content of metal incorporated in the polymers was determined by elemental analysis, AAS or ICP-OES measurements. Reaction of the deprotonated solid phase P1 with copper(II) chloride led to a blue solid-phase (P1-Cu). The UV-Vis absorption maximum of P1-Cu is found at 615 nm, which is almost identical to that found for 8. Thereby, it seems likely that P1 is flexible enough to form bisligand complexes with copper(II). This means that the copper centres act as a kind of crosslinking agents. In contrast, the heterogeneous reaction of P2 with copper(II) chloride yielded a lime green solid phase (P2-Cu). The bathochromic shift of the absorption maximum by 102 nm suggests one-sided bound copper centres.