Role of the linker region in the expression of <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> glucoamylase

Background  

Rhizopus oryzaeglucoamylase (RoGA) consists of three domains: an amino (N)-terminal raw starch-binding domain (SBD), a glycosylated linker domain, and a carboxy (C)-terminal catalytic domain. The 36-amino-acid linker region (residues 132–167) connects the two functional domains, but its structural and functional roles are unclear.     

Analysis of DNA relaxation and cleavage activities of recombinant Mycobacterium tuberculosis DNA topoisomerase I from a new expression and purification protocol

Background  

Mycobacterium tuberculosis DNA topoisomerase I is an attractive target for discovery of novel TB drugs that act by enhancing the accumulation of the topoisomerase-DNA cleavage product. It shares a common transesterification domain with other type IA DNA topoisomerases. There is, however, no homology between the C-terminal DNA binding domains of Escherichia coli and M. tuberculosis DNA topoisomerase I proteins.     

A ~35 kDa polypeptide from insect cells binds to yeast ACS like elements in the presence of ATP

Background  

The S. cerevisiae origin recognition complex binds to the ARS consensus sequence in an ATP dependent fashion. Recently, the yeast Cdc6 has been reported to have DNA binding activity. Conservation of replication proteins among different species strongly supports their functional similarity. Here we report the results of an investigation into the DNA binding activity of human Cdc6 protein. Cdc6 was expressed and purified from baculovirus infected Sf9 (Spodoptera frugiperda) insect cells as GST fusion protein (GST-Cdc6) and its DNA binding activity was tested.     

Insights into the role of Val45 and Gln182 of Escherichia coli MutY in DNA substrate binding and specificity

Background  

Escherichia coli MutY (EcMutY) reduces mutagenesis by removing adenines paired with guanines or 7,8-dihydro-8-oxo-guanines (8-oxoG). V45 and Q182 of EcMutY are considered to be the key determinants of adenine specificity. Both residues are spatially close to each other in the active site and are conserved in MutY family proteins but not in Methanobacterium thermoautotrophicum Mig.MthI T/G mismatch DNA glycosylase (A50 and L187 at the corresponding respective positions).     

Regulated interaction between polypeptide chain elongation factor-1 complex with the 26S proteasome during <Emphasis Type="Italic">Xenopus</Emphasis> oocyte maturation

Background  

During Xenopusoocyte maturation, the amount of a 48 kDa protein detected in the 26S proteasome fraction (p48) decreased markedly during oocyte maturation to the low levels seen in unfertilized eggs. The results indicate that the interaction of at least one protein with the 26S proteasome changes during oocyte maturation and early development. An alteration in proteasome function may be important for the regulation of developmental events, such as the rapid cell cycle, in the early embryo. In this study, we identified p48.     

Soluble perlecan domain i enhances vascular endothelial growth factor-165 activity and receptor phosphorylation in human bone marrow endothelial cells

Background  

Immobilized recombinant perlecan domain I (PlnDI) binds and modulates the activity of heparin-binding growth factors, in vitro. However, activities for PlnDI, in solution, have not been reported. In this study, we assessed the ability of soluble forms to modulate vascular endothelial growth factor-165 (VEGF₁₆₅) enhanced capillary tube-like formation, and VEGF receptor-2 phosphorylation of human bone marrow endothelial cells, in vitro.     

Novel acenaphthopyrazine derivatives as antitumor agents against MCF-7 cells: molecular synthesis, optical properties, and DNA-binding studies

Abstract  

A series of novel acenaphthopyrazine derivatives was synthesized from acenaphthylene-1,2-dione via three steps, including bromination, cyclization, and S_NAr^H reaction. These new compounds exhibited potential antiproliferative activity against MCF-7 cells in vitro, and 3-[2-(dimethylamino)ethylamino]acenaphtho[1,2-b]pyrazine-8,9-dicarbonitrile exhibited the highest activity (IC ₅₀ = 4.60 μM). DNA-binding experiments suggested that these derivatives bind to DNA through intercalation with intrinsic binding constants K all above 10⁵ M⁻¹. Optical property studies indicated that these compounds have long emission wavelength (λ _em > 560 nm), high quantum yields in toluene (Φ_f = 0.59 for 3-(morpholin-4-yl)acenaphtho[1,2-b]pyrazine-8,9-dicarbonitrile), and large Stokes shift (ΔS > 130 nm).     

DNA biosensors for the detection of aflatoxin producing Aspergillus flavus and A. parasiticus

Abstract  

In this work, we report on the development of a DNA-based piezoelectric biosensor specific for the detection of an amplicon of the aflD gene of Aspergillus flavus and A. parasiticus. Expression of this gene is consistently correlated with a strain’s ability to produce aflatoxins that are considered very potent liver carcinogens in various animal species and humans. The DNA biosensor has been characterized with synthetic oligonucleotides and amplicons. Moreover, it has been applied to the analysis of real samples consisting of amplicons of DNA extracted from flours and feed contaminated with A. flavus and A. parasiticus.     

Site-specific mutagenesis of <Emphasis Type="Italic">Drosophila</Emphasis> proliferating cell nuclear antigen enhances its effects on calf thymus DNA polymerase δ

Background  

We and others have shown four distinct and presumably related effects of mammalian proliferating cell nuclear antigen (PCNA) on DNA synthesis catalyzed by mammalian DNA polymerase δ(pol δ). In the presence of homologous PCNA, pol δ exhibits 1) increased absolute activity; 2) increased processivity of DNA synthesis; 3) stable binding of synthetic oligonucleotide template-primers (t_1/2 of the pol δ•PCNA•template-primer complex ≥2.5 h); and 4) enhanced synthesis of DNA opposite and beyond template base lesions. This last effect is potentially mutagenic in vivo. Biochemical studies performed in parallel with in vivo genetic analyses, would represent an extremely powerful approach to investigate further, both DNA replication and repair in eukaryotes.     

Biophysical, spectroscopic vis-à-vis biochemical investigation on DNA–metalloprotein interaction: a model study involving cobalt(II)-glutathione complex

Abstract  

The interaction of cobalt(II)-glutathione (CoGSH) with deoxyribonucleic acid (DNA) has been studied by UV–vis, fluorescence, circular dichroism (CD), thin-film infrared (IR), and viscometric techniques. From the UV-spectroscopic method, binding constant (K _b) was determined and was found to be 2.3 × 10⁶ M⁻¹. In fluorimetric analysis, the quenching of fluorescence intensity of DNA bound to ethidium bromide (EB) was investigated. The Stern–Volmer quenching constant (K _sv) was also estimated from this study and was found to be 2.8 × 10⁶ M⁻¹at 37 °C. The solution CD spectra of DNA and DNA–CoGSH indicate that in each case, DNA exists in the ‘B’ conformation and suggested an intercalative binding mode. Thin-film IR data also reveal that DNA attains the ‘B’ family of conformations after interaction with CoGSH complex. The increase in DNA viscosity in the presence of CoGSH complexes is attributed to the lengthening of DNA helix due to intercalation.     

Effects of the ancillary ligands of polypyridyl cobalt(II) complexes on pH-induced spectral properties and DNA binding properties

Abstract  

Two new Co(II) complexes [Co(ipH)₂(bdipH)]²⁺ and [Co(8-HQ)₂(bdipH)] (ipH = imidazo[4,5-f][1,10]phenanthroline, bdipH = 2-(benzo[d][1,3]dioxol-4-yl)-1H-imidazo[4,5-f][1,10]phenanthroline, 8-HQ = 8-hydroxyquinoline) were synthesized and characterized in detail by elemental analysis, IR, and UV–Vis spectroscopic techniques. The effects of pH on the UV–Vis absorption and emission spectra of the complex were studied. The interaction of the two complexes with calf thymus DNA was explored by using viscosity measurements, electronic absorption titration, competitive binding experiments, and cyclic voltammetry. The experimental results show that complex [Co(ipH)₂(bdipH)]²⁺ exhibits pH-sensitive emission, the two complexes can bind to DNA in an intercalation mode, and the DNA binding affinity of complex [Co(ipH)₂(bdipH)]²⁺ (K _b = 2.11 × 10⁵ M⁻¹) is greater than that of complex [Co(8-HQ)₂(bdipH)] (K _b = 1.76 × 10⁵ M⁻¹). The results show that the size and shape of the ancillary ligand have significant effects on the binding affinity of DNA and complexes.     

Application of chloroaluminate ionic liquid as catalyst and medium for the dealkylation of esters

Abstract  

Dealkylation of esters to carboxylic acids was performed using chloroaluminate ionic liquids (PyHBr/AlCl₃, PyHCl/AlCl₃, Me₃NHCl/AlCl₃, Et₃NHCl/AlCl₃) as catalyst and medium. The catalytic activity of PyHBr/AlCl₃ (X(AlCl₃) = 0.67) proved to be superior to the other three ionic liquids for the dealkylation of methyl benzoate with a conversion of 97% after 3 h at 140 °C. After easy separation from the products the ionic liquid PyHBr/AlCl₃ could be reused six times without loss of its activity.     

Direction of the action of glycosidases fromLimnaea stagnalis on the carbohydrate groups of the (Fc)5 region of immunoglobulins M

The action of the combined glycosidases fromL. stagnalis on the carbohydrate groups of two monoclonal immunoglobins M (Waldenström's disease) — IgM_El (I) and IgM_Ser (II) — has been studied by the semiquantitative evaluation of densitograms of glycopeptides after their separation by disc electrophoresis in polyacrylamide gel. The accessibility of the oligosaccharide groups (OGs) to the action of the glycosidases according to the positions of the OGs in a particular domain of the (Fc)₅ region of a IgM decreases for (I) in the sequence C3 C4 > hinge, and for (II) in the sequence hinge > C4 > C3. The difference in the accessibilities of the OGs (I) and (II) is apparently explained by differences in the conformations of (I) and (II). A hypothesis has been put forward concerning the possibility of the partial degradation of (II) by the accompanying proteases.N. D. Zelinskii Institute of Organic Chemistry, Academy of Sciences of the USSR, Moscow. Translated from Khimiya Prirodnykh Soedinenii, No. 3, pp. 371–375, May–June, 1984.     

A Novel Catalytic Function of Synthetic IgG‐Binding Domain (Z Domain) from Staphylococcal Protein A: Light Emission with Coelenterazine

The synthetic IgG‐binding domain (Z domain) of staphylococcal protein A catalyzes the oxidation of coelenterazine to emit light like a coelenterazine‐utilizing luciferase. The Z domain derivatives (ZZ‐gCys, Z‐gCys and Z‐domain) were purified and the luminescence properties were characterized by comparing with coelenterazine‐utilizing luciferases, including Renilla luciferase, Gaussia luciferase and the catalytic 19 kDa protein of Oplophorus luciferase. Three Z domain derivatives showed luminescence activity with coelenterazine and the order of the initial maximum intensity of luminescence was ZZ‐gCys (100%) > Z‐gCys (36.8%) > Z‐domain (1.1%) > bovine serum albumin (BSA; 0.9%) > staphylococcal protein A (0.1%) and the background value of coelenterazine (0.1%) in our conditions. The luminescence properties of ZZ‐gCys showed the similarity to that of Gaussia luciferase, including the luminescence pattern, the emission spectrum, the stimulation by halogen ions and nonionic detergents and the substrate specificity for coelenterazine analogues. In contrast, the luminescence properties of Z‐gCys were close to the catalytic 19 kDa protein of Oplophorus luciferase. The catalytic region of the Z domain for the luminescence reaction might be different from the IgG‐binding region of the Z domain.     

Synthesis of alkaloid-like compounds via the bridging Ritter reaction

Abstract  

Alkaloid-like compounds containing a benzo[c]azepine core structure were successfully prepared in three steps from 5H-dibenzo[a,d]cyclohepten-5-ol via the bridging Ritter reaction. Biological studies of these compounds revealed that some of them are AChE inhibitors and antimalarial agents.     

Calmodulin binding to recombinant myosin-1c and myosin-1c IQ peptides

Background  

Bullfrog myosin-1c contains three previously recognized calmodulin-binding IQ domains (IQ1, IQ2, and IQ3) in its neck region; we identified a fourth IQ domain (IQ4), located immediately adjacent to IQ3. How calmodulin binds to these IQ domains is the subject of this report.     

Porcine dentin sialoprotein glycosylation and glycosaminoglycan attachments

Background  

Dentin sialophosphoprotein (Dspp) is a multidomain, secreted protein that is critical for the formation of tooth dentin. Mutations in DSPP cause inherited dentin defects categorized as dentin dysplasia type II and dentinogenesis imperfecta type II and type III. Dentin sialoprotein (Dsp), the N-terminal domain of dentin sialophosphoprotein (Dspp), is a highly glycosylated proteoglycan, but little is known about the number, character, and attachment sites of its carbohydrate moieties.     

Mechanical properties and structure of the zone‐drawn poly(L‐lactic acid) fibers

The zone‐drawing (ZD) method was applied three times to the melt‐spun poly(L ‐lactic acid) (PLLA) fibers of low molecular weight (M_v = 13,100) at different temperatures under various tensions. The mechanical properties and superstructure of the ZD fibers were investigated. The resulting ZD‐3 fiber had a draw ratio of 10.5, birefringence of 37.31 × 10⁻³, and crystallinity of 37%, while an orientation factor of crystallites remarkably increased to 0.985 by the ZD‐1. The Young's modulus and tensile strength of the ZD‐3 fiber respectively attained 9.1 GPa and 275 MPa, and the dynamic storage modulus was 10.4 GPa at room temperature. © 1999 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 37: 991–996, 1999     

The reductase domain in a Type I fatty acid synthase from the apicomplexan <Emphasis Type="Italic">Cryptosporidium parvum</Emphasis>: Restricted substrate preference towards very long chain fatty acyl thioesters

Background  

The apicomplexan Cryptosporidium parvum genome possesses a 25-kb intronless open reading frame (ORF) that predicts a multifunctional Type I fatty acid synthase (CpFAS1) with at least 21 enzymatic domains. Although the architecture of CpFAS1 resembles those of bacterial polyketide synthases (PKSs), this megasynthase is predicted to function as a fatty acyl elongase as our earlier studies have indicated that the N-terminal loading unit (acyl-[ACP] ligase) prefers using intermediate to long chain fatty acids as substrates, and each of the three internal elongation modules contains a complete set of enzymes to produce a saturated fatty acyl chain. Although the activities of almost all domains were confirmed using recombinant proteins, that of the C-terminal reductase domain (CpFAS1-R) was yet undetermined. In fact, there were no published studies to report the kinetic features of any reductase domains in bacterial PKSs using purified recombinant or native proteins.