In vitro oxygen consumption and motility of Cryptobia salmositica, Cryptobia bullocki, and Cryptobia catostomi (Sarcomastigophora: Kinetoplastida).

Cryptobia salmositica (pathogenic and vaccine strains), Cryptobia bullocki (pathogenic), and Cryptobia catostomi (nonpathogenic) have similar oxygen consumption rates (0.17 +/- 0.01 nm O2/10(6) parasites). Incubation with sodium azide (5 microliters of a 1-M solution to 1 ml of parasite suspension, i.e., a 5-mM final concentration) reduced the oxygen consumption by approximately 4.5-fold. Motility of the parasites was also greatly reduced in sodium azide. The oxygen consumption and motility of the parasites returned to preazide treatment levels when the azide was removed even after 24 hr of incubation in sodium azide. The activities of hexokinase, pyruvate kinase, and cytochrome C oxidase were not detected in the 3 species of Cryptobia.     

Vector specificity of Trypanosoma catostomi and its infectivity to freshwater fishes.

Trypanosoma catostomi was found in 36.2% of 558 white suckers (Catostomus commersoni) from Ontario, Canada. The abundance of Actinobdella inequiannulata was 35% (68 leeches/197 suckers examined for leeches). The susceptibility of 3 species of leeches (Hemiclepsis marginata, Desserobdella phalera, and A. inequiannulata) and 7 species of fishes (C. commersoni, Amia calva, Anguilla rostrata, Ictalurus nebulosus, Oncorhynchus mykiss, Perca flavescens, and Esox lucius) to infection with T. catostomi was examined. Metatrypanosomes were found in the crop and proboscis sheath of 13 of 21 A. inequiannulata and in the crop of 10 of 12 H. marginata and 1 of 21 D. phalera. Only flagellates from A. inequiannulata were infective to C. commersoni. Cultured T. catostomi infected C. commersoni and A. calva but not any other fish species. Laboratory-reared C. commersoni were more susceptible than wild-caught specimens. Cultured Trypanosoma phaleri did not infect its natural host, A. calva. Host specificity should be established experimentally before a specific diagnosis is made. Cultures may be useful in simulating factors that affect development in the vector.     

Construction and characterization of two rice bacterial artificial chromosome libraries from the parents of a permanent recombinant inbred mapping population

Rice is a leading grain crop and the staple food for over half of the world population. Rice is also an ideal species for genetic and biological studies of cereal crops and other monocotyledonous plants because of its small genome and well developed genetic system. To facilitate rice genome analysis leading to physical mapping, the identification of molecular markers closely linked to economic traits, and map-based cloning, we have constructed two rice bacterial artificial chromosome (BAC) libraries from the parents of a permanent mapping population (Lemont and Teqing) consisting of 400 F9 recombinant inbred lines (RILs). Lemont (japonica) and Teqing (indica) represent the two major genomes of cultivated rice, both are leading commercial varieties and widely used germplasm in rice breeding programs. The Lemont library contains 7296 clones with an average insert size of 150 kb, which represents 2.6 rice haploid genome equivalents. The Teqing library contains 14208 clones with an average insert size of 130 kb, which represents 4.4. rice haploid genome equivalents. Three single-copy DNA probes were used to screen the libraries and at least two overlapping BAC clones were isolated with each probe from each library, ranging from 45 to 260 kb in insert size. Hybridization of BAC clones with chloroplast DNA probes and fluorescent in situ hybridization using BAC DNA as probes demonstrated that both libraries contain very few clones of chloroplast DNA origin and are likely free of chimeric clones. These data indicate that both BAC libraries should be suitable for map-based cloning of rice genes and physical mapping of the rice genome.     

Gene identification in a complex chromosomal continuum by local genomic cross-referencing

Most higher plants have complex genomes containing large quantities of repetitive DNA interspersed with low-copy-number sequences. Many of these repetitive DNAs are mobile and have homology to RNAs in various cell types. This can make it difficult to identify the genes in a long chromosomal continuum. It was decided to use genic sequence conservation and grass genome co-linearity as tools for gene identification. A bacterial artificial chromosome (BAC) clone containing sorghum genomic DNA was selected using a maize Adh1 probe. The 165 kb sorghum BAC was tested for hybridization to a set of clones representing the contiguous 280 kb of DNA flanking maize Adh1. None of the repetitive maize DNAs hybridized, but most of the low-copy-number sequences did. A low-copy-number sequence that did cross-hybridize was found to be a gene, while one that did not was found to be a low-copy-number retrotransposon that was named Reina. Regions of cross-hybridization were co-linear between the two genomes, but closer together in the smaller sorghum genome. These results indicate that local genomic cross-referencing by hybridization of orthologous clones can be an efficient and rapid technique for gene identification and studies of genome organization.     

The Best Gastric Site for Obtaining a Positive Rapid Urease Test

Background Rapid urease tests (RUTs) provide a simple, sensitive method of detecting Helicobacter pylori infection.
Objectives. Our aim, therefore, was to determine whether the yield of detecting H. pylori infection by RUT varied depending on the site of gastric biopsy.
Materials and Methods. Gastric biopsies were obtained from 50 patients for RUT by use of hp fast (GI Supply, Camp Hill, PA). Biopsies were taken from the prepyloric greater curve antrum, from the gastric angle, and from the greater curve in mid-corpus. One biopsy specimen was placed in the RUT gel, and the biopsy from the adjacent mucosa was placed in formalin for subsequent histological evaluation by using the Genta stain. RUTs were examined and scored at intervals of 5, 10, 15, 30, and 45 minutes and after 1, 2, 4, and 24 hours.
Results. Fifty patients were entered in the test (150 RUTs), 32 having H. pylori infection. There were no false-positive RUTs (specificity, 100%). The gastric angle site was positive in 100%. The prepyloric site was positive in 87%, and the corpus site was positive in 84.4% ( p < .052 for angle or prepyloric antrum versus corpus). The most common pattern was for all to be positive (74%). The median time to positivity was similar with angle and prepyloric sites (37.5 and 60 minutes, respectively, p = not significant) and shorter than the corpus biopsy (180 minutes); ( p < .05 for angle or prepyloric antrum versus corpus).
Conclusion. The maximum probability for detecting H. pylori infection using a RUT is to obtain a biopsy from the gastric angle. To prevent missing a positive result when intestinal metaplasia is present, we recommend that (at a minimum) biopsies be taken from both the angle and the corpus.     

Crystallization,molecular replacement solution,and refinement of tetrameric β-amylase from sweet potato

Sweet potato β-amylase is a tetramer of identical subunits, which are arranged to exhibit 222 molecular symmetry. Its subunit consists of 498 amino acid residues (Mr 55,880). It has been crystallized at room temperature using polyethylene glycol 1500 as precipitant. The crystals, growing to dimensions of 0.4 mm × 0.4 mm × 1.0 mm within 2 weeks, belong to the tetragonal space group P4₂2₁2 with unit cell dimensions of a = b = 129.63 Å and c = 68.42 Å. The asymmetric unit contains 1 subunit of β-amylase, with a crystal volume per protein mass (V_M) of 2.57 ų/Da and a solvent content of 52% by volume. The three-dimensional structure of the tetrameric β-amylase from sweet potato has been determined by molecular replacement methods using the monomeric structure of soybean enzyme as the starting model. The refined subunit model contains 3,863 nonhydrogen protein atoms (488 amino acid residues) and 319 water oxygen atoms. The current R-value is 20.3% for data in the resolution range of 8–2.3 Å (with 2 σ cut-off) with good stereochemistry. The subunit structure of sweet potato β-amylase (crystallized in the absence of α-cyclodextrin) is very similar to that of soybean β-amylase (complexed with α-cyclodextrin). The root-mean-square (RMS) difference for 487 equivalent Cα atoms of the two β-amylases is 0.96 Å. Each subunit of sweet potato β-amylase is composed of a large (α/β)₈ core domain, a small one made up of three long loops [L3 (residues 91–150), LA (residues 183–258), and L5 (residues 300–327)], and a long C-terminal loop formed by residues 445–493. Conserved Glu 187, believed to play an important role in catalysis, is located at the cleft between the (α/β)₈ barrel core and a small domain made up of three long loops (L3, L4, and L5). Conserved Cys 96, important in the inactivation of enzyme activity by sulfhydryl reagents, is located at the entrance of the (α/β)₈ barrel. © 1995 Wiley-Liss, Inc.     

Effect of temperature on the growth,total lipid content and fatty acid composition of recently isolated tropical microalgae Isochrysis sp., Nitzschia closterium,Nitzschia paleacea,and commercial species Isochrysis sp. (clone T.ISO)

The effect of temperature from 10 °C to 35 °C on the growth, total lipid content, and fatty acid composition of three species of tropical marine microalgae, Isochrysis sp., Nitzschia closterium, N. paleacea (formerly frustulum), and the Tahitian Isochrysis sp. (T.ISO), was investigated.Cultures of N. closterium, Isochrysis sp. and T.ISO grew very slowly at 35 °C, while N. closterium did not grow at temperatures higher than 30 °C or lower than 20 °C. N. paleacea was low-temperature tolerant, with cells growing slowly at 10 °C. N. paleacea produced the highest percentage of lipids at 10 °C, while the other species produced maximum amounts of lipid at 20 °C. None of the species maintained high levels of polyunsaturated fatty acids (PUFAs) at high growth temperature and there was a significant inverse relationship between the percentage of PUFAs and temperature for N. paleacea. A curved relationship was found between temperature and percentage of PUFA for N. closterium and tropical Isochrysis sp., with the maximum production of PUFA at 25 °C and 20 °C, respectively. The two Nitzschia species produced higher levels of the essential fatty acid eicosapentaenoic acid [20:5(n-3)] at lower growth temperatures, but the two Isochrysis species had little change in percentage of 20:5(n-3) with temperature. Only T.ISO had the highest percentage of 22:6(n-3) at lowest growth temperature (11.4% total fatty acids at 10 °C).School of Mathematical and Physical SciencesAuthor for correspondence     

Vasoactive properties of urotensins I and II in a columbiform and three galliform birds, and a bioassay for urotensin I

Summary Crude extracts ofGillichthys urophyses and chromatographically purified urotensin I (UI) and urotensin II (UII) (fromCatostomus urophyses) were injected intravenously intoCoturnix coturnix japonica, Colinus virginianus, Alectoris graeca (Galliformes) andColumba livia (Columbiformes). Changes in arterial blood pressure were monitored. UI elicited dose-dependent vasodepressor responses in all birds. Thioglycollate treatment abolished the depressor action of arginine vasotocin but not that of UI (in all birds). UII was a pressor agent inCoturnix andColinus, both members of the Galliformes. However, the hormone had no pressor activity inColumba, a member of the Columbiformes, and in another member of the Galliformes,Alectoris. The pressor effect of UII was also dose-dependent. Injection of crude urophysial extract intoColinus andCoturnix, therefore, elicited biphasic responses. UII effects can be abolished by prior incubation with carboxypeptidase A. Intravenous injection of the -adrenoceptor blocker, phenoxybenzamine, prior to urotensin injections had no effect on the responses to urotensins. As the chukar,Alectoris graeca, is sensitive to UI and resistant to anesthesia and surgery, and does not readily develop tachyphylaxis to repeated UI injections, its use is recommended as a routine bioassay animal for UI.