NaDDBS as a dispersion agent for multiwalled carbon nanotubes in capillary EKC separation of nucleotides.

A novel pseudostationary phase (PSP) of multiwalled carbon nanotubes (MWCNTs) dispersed with sodium dodecylbenzenesulfonate (NaDDBS) was used for the EKC separation of nucleotides. NaDDBS has a long hydrophobic chain and a benzylsulfonate group. It suspends more MWCNTs (about 100-fold) than SDS, and the π-π interaction between the benzene ring of NaDDBS and MWCNTs prolongs the slurry suspension time. Using NaDDBS as a surfactant can reduce the required amount of MWCNTs and decrease the baseline noise. To produce a stable suspension, the optimum ratio (w/w) of MWCNTs to NaDDBS was investigated with turbidimetry. In this context, several parameters affecting EKC separation were studied, including buffer pH, composition, concentration, and the organic modifier. Use of NaDDBS (8 mg/L)/MWCNTs (0.8 mg/L) as the PSP in a phosphate buffer (30 mM, pH 8) yielded complete resolution of seven geometric isomers of a nucleoside monophosphate. In stacking mode, with 10% MeOH in the sample plug, the mixture of nucleoside mono-, di-, and tri-phosphates was satisfactorily separated in phosphate buffer (50 mM, pH 9). The results indicate that nucleotides with bases containing more electron-withdrawing groups interact more strongly with MWCNTs. The system has been used to separate oligonucleotides, and to analyze nucleotides in a complex matrix sample.

A novel approach for rapid screening of mitochondrial D310 polymorphism.

BACKGROUND: Mutations in the mitochondrial DNA (mtDNA) have been reported in a wide variety of human neoplasms. A polynucleotide tract extending from 303 to 315 nucleotide positions (D310) within the non-coding region of mtDNA has been identified as a mutational hotspot of primary tumors. This region consists of two polycytosine stretches interrupted by a thymidine nucleotide. The number of cytosines at the first and second stretches are 7 and 5 respectively, according to the GeneBank sequence. The first stretch exhibits a polymorphic length variation (6-C to 9-C) among individuals and has been investigated in many cancer types. Large-scale studies are needed to clarify the relationship between cytosine number and cancer development/progression. However, time and money consuming methods such as radioactivity-based gel electrophoresis and sequencing, are not appropriate for the determination of this polymorphism for large case-control studies. In this study, we conducted a rapid RFLP analysis using a restriction enzyme, BsaXI, for the single step simple determination of 7-C carriers at the first stretch in D310 region. METHODS: 25 colorectal cancer patients, 25 breast cancer patients and 41 healthy individuals were enrolled into the study. PCR amplification followed by restriction enzyme digestion of D310 region was performed for RFLP analysis. Digestion products were analysed by agarose gel electrophoresis. Sequencing was also applied to samples in order to confirm the RFLP data. RESULTS: Samples containing 7-C at first stretch of D310 region were successfully determined by the BsaXI RFLP method. Heteroplasmy and homoplasmy for 7-C content was also determined as evidenced by direct sequencing. Forty-one percent of the studied samples were found to be BsaXI positive. Furthermore, BsaXI status of colorectal cancer samples were significantly different from that of healthy individuals. CONCLUSION: In conclusion, BsaXI RFLP analysis is a simple and rapid approach for the single step determination of D310 polymorphism of mitochondrial DNA. This method allows the evaluation of a significant proportion of samples without the need for sequencing- and/or radioactivity-based techniques.

Acquisition of haemoglobin-bound iron by Histophilus somni.

Histophilus somni is an important pathogen of cattle and sheep. H. somni requires iron and can use ruminant transferrins as iron sources for growth. Here, we investigated the abilities of bovine (strains 649 and 2,336) and ovine (strains 9L and 3384Y) isolates of H. somni to acquire iron from haemoglobins. Using growth assays, the bovine isolates were shown to acquire iron from bovine haemoglobin, but not from ovine, porcine or human haemoglobins; the ovine isolates, however, failed to use any of these haemoglobins as iron sources for growth. In solid phase binding assays, the bovine isolates, grown under iron-restricted conditions in the presence of bovine haemoglobin, bound not only bovine but also ovine and human haemoglobins. Competition binding assays indicated that all three haemoglobins were bound by the same receptor(s) and SDS-PAGE of membrane fractions revealed that expression of haemoglobin-binding activity was associated with the production of an approximately 120-kDa outer membrane protein. PCR approaches allowed the amplification and sequencing of hgbA, and also hugX and hugZ homologues from strains 649, 9L and 3384Y. While hgbA of strain 649 was predicted to encode an HgbA precursor that is processed to yield a mature, 123.9-kDa haemoglobin-binding protein, the hgbA genes of strains 9L and 3384Y were predicted to give rise to truncated products. RT-PCR experiments revealed that in strain 649, hugX, hugZ and hgbA are co-transcribed and iron-regulated and additional sequencing suggested that in strain 2336, expression of HgbA is subject to phase variation involving a poly C tract within hgbA.

Preparation and duplex-to-single strand transition of the fully complementary duplex of d(G4).d(C4) in aqueous solution.

The d(G4) and d(C4) molecules in the single stranded state were synthesized by the phosphotriester method and purified. The full duplex of tetramer d(G4).d(C4) was prepared by expending about a month. The duplex-to-single strand transition was observed by UV-spectroscopy. A standard hypochromic effect was observed, which is different from some experimental results reported previously.

Analysis of the secondary structure of the poly(C) tract in foot-and-mouth disease virus RNAs.

Sodium bisulphite modification of foot-and-mouth disease virus (FMDV) RNA in solution indicates that the majority of the poly(C) tract in the RNA is single-stranded in concordance with previous results with encephalomyocarditis virus RNA. The reaction kinetics are biphasic; 60% of the cytidylic acid in the poly(C) tract reacts like synthetic poly(C), and the remainder with the kinetics of the cytidylic acid in the rest of the RNA. The reactivity of the poly(C) tract with poly(I) indicates that it is looped out and exposed in the RNA. The deamination reaction has also been used to investigate the structure of the replicative form (RF) and replicative intermediate (RI) isolated from infected cells. Analysis by gel electrophoresis of the long RNase A- and T1-resistant oligonucleotides of RI suggests that it has five single-stranded poly(C) tracts to every one which is base-paired. Bisulphite reactivity of the poly(C) tract and gel electrophoresis of the ribonuclease-resistant oligonucleotides of RF indicate that the poly(C) is base-paired to a poly(G) tract in this molecule. The presence of a poly(G) tract in RF and RI provides unequivocal evidence that the poly(C) is replicated via poly(G) in the negative strand.

Neonucleoproteins. Preparation and high-performance liquid chromatographic characterization of succinyl-lysozyme-diaminooctyl-polycytidylic acid and related polycytidylic acid conjugates.

Transamination conjugates of cytidine-3'-phosphate and polycytidylic acid (poly C) with diaminopropane, diaminohexane and diaminooctane (DAO) are formed both at 25 degrees C and 60 degrees C. The extent of reaction and formation of side products, with intermittent hydrolysis to mononucleotides in the case of aminoalkyl-poly C, is monitored by reversed-phase high-performance liquid chromatography. Both Dns-DAO-poly C and succinyl-lysozyme-DAO-poly C covalent conjugates are then prepared and similarly characterized, including separation on a size-exclusion diol high-performance liquid chromatography column. The retention of the latter on a wide-pore reversed-phase column seems to be controlled by the protein moiety.

The sequence of foot-and-mouth disease virus RNA to the 5' side of the poly(C) tract.

The nucleotide sequence of foot-and-mouth disease virus (FMDV) RNA to the 5' side of the poly(C) tract (S fragment) has been determined for representatives of the A and O serotypes of the virus. The two S fragments differ in length by five nucleotides (nt), with 367 nt for O1 compared with 362 nt for A10, due to a number of insertions/deletions. However, the two sequences show 86% homology. There are no conserved open reading frames (ORFs). Secondary structure predictions reveal a high degree of potential base-pairing, such that the entire S fragment sequence can be folded into a hairpin structure.