Determination of desmosine, isodesmosine, and other amino acids by liquid chromatography with electrochemical detection following precolumn derivatization with naphthalenedialdehyde/cyanide

Naphthalenedialdehyde (NDA) in the presence of cyanide (CN) reacts with primary amines to produce fluorescent cyano[f]benzoisoindole (CBI) derivatives. These derivatives have been shown to be substantially more stable than the corresponding o-phthalaldehyde derivatives. However, one drawback of this method is that compounds derivatized at more than one site exhibit quenching, precluding the use of fluorescence detection. The CBI derivatives have been found to be electroactive and are oxidized at a modest oxidation potential (+750 mV). Electrochemical detection is especially useful for the analysis of compounds containing more than one primary amine site because the response is not attenuated as it is in fluorescence detection. Desmosine and isodesmosine were of particular interest because of their importance in elastic fiber and the lack of highly sensitive HPLC methods for the determination of these compounds. Both of these compounds react with NDA/CN to produce electrochemically active derivatives. The combination of derivatization with NDA/CN and electrochemical detection was found to be linear over three orders of magnitude. Detection limits for CBI-lysine and CBI-desmosine were 100 fmol at a S/N of 2. Amino acids in elastin were quantitated using this method. The results correlate well with what has been reported previously in the literature. A significant advantage of the use of liquid chromatography with electrochemical detection with precolumn derivatization with NDA/CN for the analysis of desmosine and isodesmosine is that they can be separated and quantitated individually using this method. In addition, the unique voltammetry of multiderivatized CBI-amino acids can be used to verify peak purity.     

Chemotaxis away from thiocyanic and isothiocyanic esters in Pseudomonas aeruginosa

Abstract A novel mycoplasmal species designated as Mycoplasma penetrans has recently been isolated from patients infected with human immunodeficiency virus. The 16S rRNA gene from this mycoplasma was cloned and its nucleotide sequence determined. This sequence was aligned with previously published homologous sequences from several mycoplasmas and with related Gram-positive bacteria and a phylogenetic tree was constructed. The results indicate that M. penetrans belongs to the evolutionary group Pneumoniae.     

Cloning and nucleotide sequence of fosfomycin biosynthetic genes of Streptomyces wedmorensis

The biosynthetic pathway for production of the antibiotic fosfomycin by Streptomyces wedmorensis consists of four steps including the formation of a C-P bond and an epoxide. Fosfomycin production genes were cloned from genomic DNA using S. wedmorensis mutants blocked at different steps of the biosynthetic pathway. Four genes corresponding to each of the biosynthetic steps were found to be clustered in a DNA fragment of about 5 kb. Nucleotide sequencing of a large fragment revealed the presence of ten open reading frames, including the four biosynthetic genes and six genes with unknown functions.     

Gravitropic reaction of primary seminal roots ofZea mays L. influenced by temperature and soil water potential

The growth of the primary seminal root of maize (Zea mays L.) is characterized by an initial negative gravitropic reaction and a later positive one that attains a plagiotropic liminal angle. The effects of temperature and water potential of the surrounding soil on these gravitropic reactions were studied. Temperatures of 32, 25, and 18C and soil water potentials of −5,−38, and −67 kPa were imposed and the direction of growth was measured for every 1 cm length of the root. The initial negative gravitropic reaction extended to a distance of about 10cm from the graln. Higher temperatures reduced the initial negative gravitropic reaction. Lower soil water potential induced a downward growth at root emergence. A mathematical model, in which it was assumed that the rate of the directional change of root growth was a sum of a time-dependent negative gravitropic reaction and an establishment of the liminal angle, adequately fitted the distance-angle relations. It was suggested that higher temperatures and/or a lower water potential accelerated the diminution of the intitial negative gravitropic reaction.     

A basal unit of valine-sensitive acetolactate synthase of Neurospora crassa

Valine-sensitivity as well as activity of acetolactate synthase of Neurospora crassa was stabilized with 1.2 M potassium phosphate buffer during extraction from mitochondria and early stages of purification, and with 20% glycerol plus 5 mM sodium pyruvate during Sephadex G200 gel chromatography. The enzyme was expressed as four molecular species having the molecular weights of about 500,000, 140,000, 68,000 and 51,000, respectively. The first and the third species showed valine-sensitivity, but the second and the fourth did not. The third molecular species with a molecular weight of 68,000 may be the basal unit of valine-sensitive acetolactate synthase of Neurospora crassa.     

Mapping quantitative trait loci for root development under hypoxia conditions in soybean (<Emphasis Type="Italic">Glycine max</Emphasis> L. Merr.)

Key message

Greatest potential, QTLs for hypoxia and waterlogging tolerance in soybean roots were detected using a new phenotypic evaluation method.

Abstract

Waterlogging is a major environmental stress limiting soybean yield in wet parts of the world. Root development is an important indicator of hypoxia tolerance in soybean. However, little is known about the genetic control of root development under hypoxia. This study was conducted to identify quantitative trait loci (QTLs) responsible for root development under hypoxia. Recombinant inbred lines (RILs) developed from a cross between a hypoxia-sensitive cultivar, Tachinagaha, and a tolerant landrace, Iyodaizu, were used. Seedlings were subjected to hypoxia, and root development was evaluated with the value change in root traits between after and before treatments. We found 230 polymorphic markers spanning 2519.2 cM distributed on all 20 chromosomes (Chrs.). Using these, we found 11 QTLs for root length (RL), root length development (RLD), root surface area (RSA), root surface area development (RSAD), root diameter (RD), and change in average root diameter (CARD) on Chrs. 11, 12, 13 and 14, and 7 QTLs for hypoxia tolerance of these root traits. These included QTLs for RLD and RSAD between markers Satt052 and Satt302 on Chr. 12, which are important markers of hypoxia tolerance in soybean; those QTLs were stable between 2 years. To validate the QTLs, we developed a near-isogenic line with the QTL region derived from Iyodaizu. The line performed well under both hypoxia and waterlogging, suggesting that the region contains one or more genes with large effects on root development. These findings may be useful for fine mapping and positional cloning of gene responsible for root development under hypoxia.

     

Acute exercise increases expression of extracellular superoxide dismutase in skeletal muscle and the aorta

Exercise dramatically increases oxygen consumption and causes oxidative stress. Superoxide dismutase (SOD) is important in the first-line defence mechanisms against oxidative stress. To investigate the effect of acute exercise on the expression of SOD, we examined the expression of mRNA for three SOD isozymes, in mice run on a treadmill to exhaustion. Six hours after exercise, the expression of extracellular SOD (EC-SOD) mRNA increased significantly in skeletal muscle and persisted for 24 h, whereas no change was observed for cytoplasmic and mitochondrial SOD mRNA. Moreover, acute exercise also induced EC-SOD mRNA in the aorta. These results suggest that a single bout of exercise is enough to augment the expression EC-SOD mRNA in skeletal muscle and the aorta, and may partly explain the beneficial effect of exercise.     

High-performance liquid chromatographic determination of short-chain aliphatic aldehydes using 4-(N,N-dimethylaminosulphonyl)-7-hydrazino-2,1,3-benzoxadiazole as a fluorescence reagent

High-performance liquid chromatographic determination of four short-chain aliphatic aldehydes using fluorescence detection was carried out with 4-(N,N-dimethylaminosulphonyl)-7-hydrazino-2,1,3-benzoxadiazole (DBD-H). DBD-H derivatives with three aliphatic aldehydes — formaldehyde, acetaldehyde and propionaldehyde — were synthesized and their fluorescence properties were examined. Relative fluorescence intensities of these compounds in acetonitrile were ca. ten-fold larger than those in aqueous acetonitrile. DBD-hydrazones could be separated by reversed-phase chromatography using aqueous acetonitrile as eluent and detection at 560 nm with excitation at 445 nm. Submicromolar levels of formaldehyde, acetaldehyde, propionaldehyde and butylaldehyde could be determined. The HPLC procedure using propionaldehyde as internal standard was applied to the measurement of acetaldehyde levels in normal human plasma before and 30 min after ingestion of ethanol.     

Possible involvement of Hcn1 ion channel in learning and memory dysfunction in SAMP8 mice

The senescence-accelerated mouse prone 8 (SAMP8) strain exhibits age-related learning and memory deficits (LMD) at 2 months of age. Combined linkage analysis of 264 F2 intercross SAMP8 × JF1 mice and RNA-seq analysis identified Hcn1 gene out of 29 genes in the LMD region on chromosome 13. Hcn1 in SAMP8 strain showed 15 times less polyglutamine repetition compared to Japanese fancy mouse 1 (JF1). Whole cell patch clamp analysis showed that Hcn1 ion conductivity was significantly lower in SAMP8 compared to that of JF1, which may be associated with learning and memory deficiency.