Eimeria tenella,E. necatrix,E. acervulina,and E. maxima: Anticoccidial activity of 1,6-dihydro-6-oxo-2-pyrazinecarboxylic acid 4-oxide

The anticoccidial activity of an orotic acid analog, 1,6-dihydro-6-oxo-2-pyrazinecarboxylic acid 4-oxide (carboxyemimycin), was tested in battery experiments, utilizing 9-day-old Single-Comb White Leghorn cockerels. Carboxyemimycin, at 125 ppm and more in feed, exhibited marked anticoccidial activities against Eimeria tenella, E. necatrix, E. acervulina, and E. maxima. High doses of carboxyemimycin—up to 1000 ppm—did not cause any reduction in weight gains. The battery and in vitro studies with delayed and restricted medications revealed that carboxyemimycin affected the development of E. tenella in first and second generation schizogony and in gametogony.     

Chemical Synthesis of Oligonucleotides Containing The (6-4) Photoproduct at the Thymine-Cytosine Site and Its Repair By (6-4) Photolyase

Abstract

A phosphoramidite building block of the T(6-4)C photoproduct was synthesized. One of the differences from T(6-4)T was formation of cytosine hydrates by UV irradiation, and the other was acylation of the amino function with the capping reagent. The capping step was omitted to improve the yield of the desired oligonucleotides. Characterization of the (6-4) photolyase using one of the oligonucleotides revealed that this enzyme restores the pyrimidines in T(6-4)C to their original structures.     

Purification of an β-D-(1→3)-Glucanase from Rhizopus niveus

An β-D-(l→3)-glucanase has been purified from the culture medium of Rhizopus niveus. The purification involves calcium acetate treatment, polyethylene glycol 6000 fractionation, CM-cellulose batch treatment, DEAE-cellulose column chromatography and gel filtration on Sephadex G–150.

The final preparation is homogenous on the basis of discelectrophoresis on acryl amide gel, sedimentation in the ultracentrifuge.

Some properties of the purified enzyme have been also tested.     

Structural study of DNA duplexes containing the (6–4) photoproduct by fluorescence resonance energy transfer

Fluorescence resonance energy transfer (FRET) experiments have been performed to elucidate the structural features of oligonucleotide duplexes containing the pyrimidine(6–4)pyrimidone photoproduct, which is one of the major DNA lesions formed at dipyrimidine sites by UV light. Synthetic 32mer duplexes with and without the (6–4) photoproduct were prepared and fluorescein and tetramethylrhodamine were attached, as a donor and an acceptor, respectively, to the aminohexyl linker at the C5 position of thymine in each strand. Steady-state and time-resolved analyses revealed that both the FRET efficiency and the fluorescence lifetime of the duplex containing the (6–4) photoproduct were almost identical to those of the undamaged duplex, while marked differences were observed for a cisplatin-modified duplex, as a model of kinked DNA. Lifetime measurements of a series of duplexes containing the (6–4) photoproduct, in which the fluorescein position was changed systematically, revealed a small unwinding at the damage site, but did not suggest a kinked structure. These results indicate that formation of the (6–4) photoproduct induces only a small change in the DNA structure, in contrast to the large kink at the (6–4) photoproduct site reported in an NMR study.     

A critical role of the 3' terminus of nascent DNA chains in recognition of stalled replication forks

Arrest of replication forks by various internal and external threats evokes a myriad of cellular reactions, collectively known as DNA replication checkpoint responses. In bacteria, PriA is essential for restoration of stalled replication forks and recombinational repair of double-stranded DNA breaks and is a candidate sensor protein that may recognize arrested forks. Here, we report that PriA protein specifically recognizes 3' termini of arrested nascent DNA chains at model stalled replication forks in vitro. Mutations in the putative "3' terminus binding pocket" present in the N-terminal segment of PriA result in reduced binding to stalled replication fork structures and loss of its biological functions. The results suggest a mechanism by which stalled replication forks are recognized by a sensor protein for checkpoint responses.     

Complete nucleotide sequence of the S10-spc operon of phytoplasma: gene organization and genetic code resemble those of Bacillus subtilis

An 11.4-kbp region of genomic DNA containing the complete S10-spc operon was constructed by an integrative mapping technique with eight plasmid vectors carrying ribosomal protein sequences from onion yellows phytoplasma. Southern hybridization analysis indicated that phytoplasmal S10-spc is a single-copy operon. This is the first complete S10-spc operon of a phytoplasma to be reported, although only a part of six serial genes of the S10 operon is reported previously. The operon has a context of 5'-rps10, rpl3, rpl4, rpl23, rpl2, rps19, rpl22, rps3, rpl16, rpl29, rps17, rpl14, rpl24, rpl5, rps14, rps8, rpl6, rpl18, rps5, rpl30, rpl15, SecY-3', and is composed of 21 ribosomal protein subunit genes and a SecY protein translocase subunit gene. Resembling Bacillus, this operon contains an rpl30 gene that other mollicutes (Mycoplasma genitalium, M. pneumoniae, and M. pulmonis) lack. A phylogenetic tree based on the rps3 sequence showed that phytoplasmas are phylogenetically closer to acholeplasmas and bacillus than to mycoplasmas. In the S10-spc operon, translation may start from either a GTG codon or an ATG codon, and stop at a TGA codon, as has been reported for acholeplasmas and bacillus. However, in mycoplasmas, GTG was found as a start codon, and TGA was found not as a stop codon, but instead as a tryptophan codon. These data derived from the gene organization, and the genetic code deviation support the hypothesis that phytoplasmal genes resemble those of acholeplasmas and Bacillus more than those of other mollicutes.     

Synthesis of CMP-9'-modified-sialic acids as donor substrate analogues for mammalian and bacterial sialyltransferases

Cytidine-5'-monophospho-sialic acid (CMP-Neu5Ac) derivatives bearing a phenyl group in which the tether length between the phenyl group and the 9-position of Neu5Ac varied were synthesized and evaluated as substrates for sialyltransferases. In the synthesis of the compounds, a coupling reaction between methyl 5-acetamido-4,7,8-tri-O-acetyl-9-azido-3,5,9-trideoxy-beta-D-glycero-D-galacto-2-nonulopyranosonate and 2-cyanoethyl 2',3'-O,N4, triacetylcytidine-5'-yl N,N-diisopropylphosphoramidite was carried out and the phosphite derivative thus obtained was oxidized and then deprotected to yield CMP-9'-azido-Neu5Ac. Modification of the 9-amino group prepared by reduction of the azido groups was performed by the use of several phenyl-substituted alkylcarboxylic acid derivatives. Using these CMP-9'-modified-Neu5Ac analogues bearing the phenyl-substituted alkyl-amide group, sialyltransferase assays were performed with both rat liver alpha-(2-->6)-sialyltransferase and Photobacterium alpha-(2-->6)-sialyltransferase. These 9-modified analogues could be transferred to disaccharide acceptors, and a practical enzymatic synthesis using CMP-9'-modified-Neu5Ac yielded sialoside analogues and sialylglycoproteins in good yield. These experiments demonstrate that the Photobacterium sialyltransferase can be used in the synthesis of sialoside analogues having a large substituent at the 9-position of Neu5Ac.     

DNA detection system using molecularly imprinted polymer as the gel matrix in electrophoresis

To develop a simple and inexpensive method for DNA detection, we prepared a molecularly imprinted polymer (MIP) for recognizing a specific double-stranded DNA (dsDNA) sequence and used it in an electrophoretic gel matrix. The MIP gel has many binding sites that are complementary in size, shape, and arrangement of functional groups of the target dsDNA sequence. During MIP gel electrophoresis (MIPGE), migration of the target dsDNA should be hindered by the capture effect of the binding sites in the MIP gel. This was confirmed by observation of deviations from the linear relationship between the migration distances of the DNA standard size markers in the polyacrylamide gel and those in the MIP gel. The migration distances of nontarget dsDNA maintained a linear relationship, however. In addition, the sequence selectivity of dsDNA in this method was investigated by using the Ha-ras gene and its point mutants. Except for A.T to T.A base pair substitution, mutant dsDNA (for example, substitution from A.T to C.G and from G.C to T.A) could be distinguished from the target (wild-type) dsDNA. Although some improvement in A.T (T.A) base pair distinction is still needed, this study is the first to demonstrate detection of a specific dsDNA sequence with MIPs and, as such, opens up a new realm for practical applications of MIPs.