Simultaneous determination of histamine and N tau-methylhistamine with high-performance liquid chromatography using electrochemical detection

We have developed a liquid chromatographic method which uses electrochemical detection for the simultaneous quantitation of histamine and N tau-methylhistamine in rat brain. The amines are derivatized with the water-soluble Bolton-Hunter reagent (sulfo B-H). Perchloric acid extracts of rat brains are chromatographed on a strong cation-exchange resin. The eluate is evaporated and allowed to react with sulfo B-H at pH 9.8 at room temperature. The derivatization is complete after 30 s vortexing. The derivatives are purified using a cellulose-phosphate fibrous cation exchanger. They are quantified with an electrochemical detector at a potential of 0.56 V after preoxidizing the sample at 0.47 V. The derivatives of histamine, N tau-methylhistamine, and N alpha-methylhistamine are completely separated without interfering peaks. Since no N alpha-methylhistamine was detected in rat brain it was used as an internal standard. The detection limits are 0.1 pmol of histamine and 0.2 pmol of N tau-methylhistamine. The precision of this method is high, with within-run and between-run coefficients of variation of 2-7% and linearity of 0.999. Both histamine and N tau-methylhistamine peak heights increased significantly and selectively after treatment with pargyline. Because of the high sensitivity, accuracy, and precision, the histamine and N tau-methylhistamine contents of single nuclei of the rat hypothalamus can be routinely quantified.     

Pesticide formulations: Innovations and developments

Book Review

Pesticide formulations: Innovations and developmentsB. Cross and H.B. Scher (Eds.), ACS Symposium Series 371. Washington DC: American Chemical Society, 1988. xi + 288 pages. £54.00. ISBN 0-8412-1483-2     

Dissolved organic carbon concentration of a natural water body and its relationship to water color in Central Kalimantan, Indonesia

In Central Kalimantan, Indonesia, the water of lakes and rivers showed high dissolved organic carbon (DOC) concentration, 5-50mgC/l. There was a clear relationship between DOC concentration and pH. DOC also contributed to low water transparency in the studied lakes. Water colors measured by spectrophotometer showed a strong relationship with DOC concentration, indicating high applicability of water color measurement for estimation of DOC. The development of a simple but quick estimation of DOC will contribute to understanding the seasonal dynamics of DOC, which might regulate both abiotic and biotic conditions in aquatic ecosystems in this area.     

Five Cyanophora (Cyanophorales,Glaucophyta) species delineated based on morphological and molecular data

Cyanophora is an important glaucophyte genus of unicellular biflagellates that may have retained ancestral features of photosynthetic eukaryotes. The nuclear genome of Cyanophora was recently sequenced, but taxonomic studies of more than two strains are lacking for this genus. Furthermore, no study has used molecular methods to taxonomically delineate Cyanophora species. Here, we delimited the species of Cyanophora using light and electron microscopy, combined with molecular data from several globally distributed strains, including one newly established. Using a light microscope, we identified two distinct morphological groups: one with ovoid to ellipsoidal vegetative cells and another with dorsoventrally flattened or broad, bean‐shaped vegetative cells containing duplicated plastids. Our light and scanning electron microscopy clearly distinguished three species with ovoid to ellipsoidal cells (C. paradoxa Korshikov, C. cuspidata Tos.Takah. & Nozaki sp. nov., and C. kugrensii Tos.Takah. & Nozaki sp. nov.) and two species with broad, bean‐shaped cells (C. biloba Kugrens, B.L.Clay, C.J.Mey. & R.E.Lee and C. sudae Tos.Takah. & Nozaki sp. nov.) based on differences in cell shape and surface ornamentations of the vegetative cells under the field‐emission scanning electron microscope. Molecular phylogenetic analyses of P700 chl a apoprotein A2 (psaB) genes and internal transcribed spacer (ITS) regions of nuclear ribosomal DNA (rDNA), as well as a comparison of secondary structures of nuclear rDNA ITS‐2 and genetic distances of psaB genes, supported the delineation of five morphological species of Cyanophora.     

Species richness and species composition of fungal communities associated with cellulose decomposition at different altitudes on the Tibetan Plateau

Aims The aims of this study were to compare the fungal communities developing on cotton strips at three different altitudes on the Tibetan Plateau and to assess the environmental variables influencing them.Methods Cotton strips that had been buried in soil for a year were sampled at three sites at different altitudes (4500, 4950 and 5200 m) located on a southeast-facing slope on the Nyainqentanglha Mountains near Damxung. The fungi on the cotton strips were isolated using a modified washing method. The decomposition abilities and colony growth properties of the major species cultured in pure-culture conditions were investigated and compared. Canonical correspondence analysis (CCA) was used to evaluate the relationships between fungal community composition and environmental variables (altitude, soil depth, soil water content [SWC], plant root mass and gravel content).Important findings A total of 24 species were isolated from the cotton strips, and 12 species occurred frequently and were regarded as major species. The number of fungal species was lower at the 4950-m altitude site than at the other two sites, indicating that not only altitude but also other factors affected the number of species present. All of the major species were able to decompose the cotton strips. In the CCA ordination, automatic forward selection revealed that altitude, SWC and plant root mass significantly affected fungal species composition. Our results suggest that species number and the composition of cellulolytic fungal communities are highly correlated with environmental variables as well as altitude in the alpine meadow on the Tibetan Plateau.     

Possible Host Adaptation as an Evolution Factor of Cowpea aphid‐borne mosaic virus Deduced by Coat Protein Gene Analysis

Cowpea aphid‐borne mosaic virus (CABMV) causes major diseases in cowpea and passion flower plants in Brazil and also in other countries. CABMV has also been isolated from leguminous species including, Cassia hoffmannseggii, Canavalia rosea, Crotalaria juncea and Arachis hypogaea in Brazil. The virus seems to be adapted to two distinct families, the Passifloraceae and Fabaceae. Aiming to identify CABMV and elucidate a possible host adaptation of this virus species, isolates from cowpea, passion flower and C. hoffmannseggii collected in the states of Pernambuco and Rio Grande do Norte were analysed by sequencing the complete coat protein genes. A phylogenetic tree was constructed based on the obtained sequences and those available in public databases. Major Brazilian isolates from passion flower, independently of the geographical distances among them, were grouped in three different clusters. The possible host adaptation was also observed in fabaceous‐infecting CABMV Brazilian isolates. These host adaptations possibly occurred independently within Brazil, so all these clusters belong to a bigger Brazilian cluster. Nevertheless, African passion flower or cowpea‐infecting isolates formed totally different clusters. These results showed that host adaptation could be one factor for CABMV evolution, although geographical isolation is a stronger factor.     

Crystallization and Preliminary X-Ray Analysis of Neuropsin, a Serine Protease Expressed in the Limbic System of Mouse Brain

Neuropsin (M_r25 032) is a serine protease expressed in the limbic system of mouse brain. It has been implicated in various neurological processes including formation of memory and may be important as a drug target in the treatment of epilepsy. The recombinant protein was produced using a baculovirus expression system and was purified. Two crystal forms were obtained by a hanging-drop vapor-diffusion method with polyethylene glycol. Preliminary X-ray crystallographic analysis revealed that crystal form I belongs to triclinic space groupP1 with unit cell dimensionsa= 97.16 Å,b= 97.12 Å,c= 46.75 Å and α = 99.17°, β = 99.77°, γ = 117.35°. Self-rotation function analysis of these data of form I indicates the position of a noncrystallographic threefold axis. There are six molecules in the crystallographic asymmetric unit. Crystal form II also belongs to triclinic space groupP1 but has unit cell dimensions ofa= 38.40 Å,b= 55.16 Å,c= 65.37 Å and α = 95.38°, β = 89.98°, γ = 110.46° with two molecules in the crystallographic asymmetric unit. Form II has a noncrystallographic twofold axis. Intensity data to 3.1 Å resolution for form I and to 2.2 Å resolution for form II have been collected.     

Establishment of a SPF colony of human apo(a) transgenic rabbits by frozen-thawed embryo transfer.

In the present study, embryo transfer was performed using frozen-thawed embryos to establish a SPF colony of human apolipoprotein (a) (apo(a)) transgenic rabbits. Apo(a) transgenic rabbits were kept under conventional condition and were infected with Bordetella bronchiseptica. Embryos at the morula stage were collected and stored in liquid nitrogen. After thawing, the in vitro survival rate was 84.6%, and 125 morphologically normal embryos were transferred to 6 SPF recipient rabbits. Four rabbits became pregnant and 23 live pups were born. PCR and Western blot analyses revealed that 9 of 23 pups were transgenic and expressed apo(a) protein. Microbiological tests showed all rabbits were free from infections. We succeeded in establishing a SPF colony of apo(a) transgenic rabbits. These rabbits are now maintained under a barrier system and are available for medical research.     

Cytokine regulation of cell-to-cell interactions in lymphokine-activated killer cell cytotoxicity in vitro

The permanent pancreas carcinoma cell line, PCI-24, was developed in order to analyse cytokine regulation on pancreas carcinoma and lymphokine-activated killer (LAK) cell interaction. PCI cells expressed ICAM-1 and HLA-ABC, but not HLA-DR antigens. PCI cells showed augmented ICAM-1 and HLA-ABC expression when incubated with interferon (IFN) and tumour necrosis factor . A similar but weak augmentary effect on the HLA-ABC and ICAM-1 surface expression was seen with interleukin-1 treatment. Natural attachment of LAK to PCI cells was augmented by recombinant IFN in close association with ICAM-1 up-regulation on PCI cells. In addition, natural attachment was significantly inhibited by anti-LFA-1 and anti-ICAM-1 antibody treatments. Cytotoxicity of the LAK cells against PCI cells was also significantly inhibited with the same treatment. Thus, the attachment of LAK cells to PCI cells through LFA-1/ICAM-1 molecules appeared to be essential for the cytotoxicity for PCI cells. Pretreatment of PCI cells, but not of LAK cells, with IFN or other cytokines resulted in a decrease of susceptibility for LAK cell cytotoxicity. The decreased susceptibility inversely correlated with HLA-ABC expression on the PCI cells. The collective evidence indicates that, although LAK cell attachment to pancreas carcinoma cells through the LFA-1/ICAM-1 molecule is augmented by IFN, IFN treatment of pancreas carcinoma cells reduces LAK cell cytotoxicity possibly through an increase in HLA-ABC or a regulation of molecules closely associated to HLA-ABC expression.