HALOGENATED MONOTERPENE PRODUCTION IN REGENERATED PLANTLET CULTURES OF OCHTODES SECUNDIRAMEA (RHODOPHYTA, CRYPTONEMIALES)

Three genera of macrophytic red algae ( Ochtodes , Plocamium , and Portieria ) contain novel halogenated monoterpenes. To develop an in vitro system for studying halogenated monoterpene production, a laboratory tissue culture was established for Ochtodes secundiramea (Montagne) Howe (Cryptonemiales, Rhizophyllidaceae). Specifically, callus cells were induced from thallus explants of O. secundiramea plants. Shoot primordia regenerated from callus cells and developed into plantlets that released tetraspores. A sporeling from one of these tetraspores was selected for further culture. Axenic plantlets were cultivated in ESS-enriched natural seawater. Thallus tissue was cut into small pieces before subculture. Each plantlet grew as a symmetrical array of highly branched shoot tissues emanating from a common center, ultimately assuming a spherical shape of 20 mm diameter 4 weeks after subculture. Specific growth rates of over 20% per day were attained in bubble-aerated flask culture at an optimal temperature of 26° C and photosynthetic saturation light intensity of 200 μmol photons·m ^{− 2}·s ^{− 1}. The cultured plantlets contained myrcene and seven halogenated monoterpenes, based on gas chromatography–mass spectroscopy analysis of dichloromethane extracts. Although bromomyrcene was the dominant acyclic halogenated monoterpene, the cyclic halogenated monoterpenes chondrocole C and ochtodene were also produced by the O. secundiramea plantlet cultures. Halogenated monoterpene production at light-saturated growth conditions increased with decreasing nitrogen availability below 1.0 mM medium nitrate concentration (N:P ratios of 1.6:1 to 32:1). The halogenated monoterpene yield was insensitive to medium nitrate concentrations above 1.0 mM (N:P ratios of 32:1 to 320:1), where the bromomyrcene yield was 1700 μg per gram of dry cell mass.     

Development and testing of a bovine trichomoniasis vaccine

Eighteen culled dairy cows were randomly allocated into 1 of 5 treatment groups. Six cows were vaccinated twice (2V), 21 days apart, 3 with whole cell (2WC) and 3 with fragmented cell membrane (2FC) containing 1 x 10(9)Trichomonas fetus organisms or protein equivalent in a commercial mineral oil adjuvant vaccine. Six more cows were vaccinated once (1V), 3 with whole cell (1WC) and 3 with fragmented cell vaccine (1FC), using the same vaccine, while 6 cows were used as the unvaccinated controls. All cows were challenged with 1 x 10(5) organisms 4 weeks after the second or the only vaccination. After challenge, cervico-vaginal mucus (CVM) samples were cultured for T . fetus weekly for 9 weeks. Whole cell vaccines were superior to fragmented cell vaccines, and both performed better than no vaccination for apparent elimination of trichomonad infections in dairy cows. In addition, 2V was superior to 1V, which, in turn, was superior to no vaccination. Furthermore, clearance time was reduced most by 2V and whole cell vaccination compared with 1V and fragmented cell vaccination. Clearance time was decreased significantly in all vaccinated cows compared with that in unvaccinated cows.     

Bovine trichomoniasis: Results of a slaughter survey in Colorado and Nebraska

A total of 2,909 bulls was cultured for the presence of Trichomonas fetus at slaughter at 4 abattoirs in Colorado and Nebraska. Samples were collected using the dry pipette preputial scraping technique and were transported to diagnostic laboratories within 24 h. Of the bulls cultured, 5 were positive. The state of the last origin for each animal was Kansas, 1 bull, Montana, 1 bull, Nebraska, 1 bull, and Utah, 2 bulls. These results indicate and incidence of less than 1% (0.172%) of trichomoniasis in the populations tested, even in the states of Colorado and Nebraska where the larger numbers of bulls were cultured.     

Drosophila rab GDI mutants disrupt development but have normal rab membrane extraction

Summary: Rab GTPases are essential for vesicular transport. Rab GDP dissociation inhibitor (GDI) binds to GDP‐bound rabs, removes rabs from acceptor membranes and delivers rabs to donor membranes. We isolated lethal GDI mutations in Drosophila and analyzed their developmental phenotypes. To learn how these mutations affect GDI structure, the crystal structure of Drosophila GDI was determined by molecular replacement to a resolution of 3.0 Å. Two hypomorphic, missense mutations are located in domain II of GDI at highly conserved positions, but not in previously identified sequence conserved regions. The mutant GDIs were tested for ability to extract rabs from membranes and showed wild‐type levels of rab membrane extraction. The two missense alleles showed intragenic complementation, indicating that domain II of GDI may have two separable functions. This study indicates that GDI function is essential for development of a complex, multicellular organism and that puparium formation and pole cell formation are especially dependent on GDI function. genesis 31:17–29, 2001. © 2001 Wiley‐Liss, Inc.     

Analysis of the human Alu Ya-lineage

The Alu Ya-lineage is a group of related, short interspersed elements (SINEs) found in primates. This lineage includes subfamilies Ya1-Ya5, Ya5a2 and others. Some of these subfamilies are still actively mobilizing in the human genome. We have analyzed 2482 elements that reside in the human genome draft sequence and focused our analyses on the 2318 human autosomal Ya Alu elements. A total of 1470 autosomal loci were subjected to polymerase chain reaction (PCR)-based assays that allow analysis of individual Ya-lineage Alu elements. About 22% (313/1452) of the Ya-lineage Alu elements were polymorphic for the insertion presence on human autosomes. Less than 0.01% (5/1452) of the Ya-lineage loci analyzed displayed insertions in orthologous loci in non-human primate genomes. DNA sequence analysis of the orthologous inserts showed that the orthologous loci contained older pre-existing Y, Sc or Sq Alu subfamily elements that were the result of parallel forward insertions or involved in gene conversion events in the human lineage. This study is the largest analysis of a group of "young", evolutionarily related human subfamilies. The size, evolutionary age and variable allele insertion frequencies of several of these subfamilies makes members of the Ya-lineage useful tools for human population studies and primate phylogenetics.     

Relationships of microsporidian genera, with emphasis on the polysporous genera, revealed by sequences of the largest subunit of RNA polymerase II (RPB1)

Molecular data have proved useful as an alternative to morphological data in showing the relationships of genera within the phylum Microsporidia, but until now have been available only for ribosomal genes. In previous studies protein-coding genes of microsporidia have been used only to assess their position in the evolution of eukaryotes. For the first time we report on the use of a protein-coding gene, the A-G region of the largest subunit of RNA polymerase II (RPB1) from 14 mainly polysporous species, to generate an alternative phylogeny for microsporidia. Using the amino acid sequences, the genera and species fell into the same main groupings as had been obtained with 16S rDNA sequences, but the RPB1 data provided better resolution within these groups. The results supported the pairings of Trachipleistophora hominis with Vavraia culicis and Pleistophora hippoglossoideos with Pleistophora typicalis. They also confirmed that the genus Pleistophora is not monophyletic and that it will be necessary to transfer Pleistophora ovariae and Pleistophora mirandellae into one or more other genera, as has already been effected for Pleistophora anguillarum.     

Prophylaxis by vitamin C in starvation induced rat stomach ulceration

Rats given L-ascorbic acid in their drinking water prior to and during starvation did not develop severe ulceration in the mucosal lining of their stomachs. Control rats which were either nontreated or given deactivated L-ascorbic acid developed severe stomach pathology on the starvation regimen.     

Possible defect in cholinergic neurons of muscular dystrophic mice.

The cholinoacetyltransferase activity (CAT) in diaphragm of mice of Bar Harbor strain (129 ReJ dy/dy) with muscular dystrophy was significantly lower than that of phenotypically normal litter mates (129 ReJ dy/+). CAT, tyrosine hydroxylase (TH), dopamine-β-hydroxylase (DβH) activities were found identical in adrenal gland and brain homogenates of normal and dystrophic mice. Subacute injections of atropine (72 μmol/kg i. p., twice daily for 3 days) failed to increase the activity of adrenal CAT in dystrophic mice but increased this enzyme activity in adrenals of normal litter mates. The concentration in brain of dopamine, norepinephrine, serotonin, acetylcholine (ACh), γ-aminobutyric acid (GABA) and some of their precursors were measured. Only the concentration of ACh was significantly lower in the brain of muscular dystrophic mice. The rate of accumulation of brain ACh concentration after the injection of oxotremorine (5μmol/kg i. p.) is slower in muscular dystrophic animals than in normal litter mates. Furthermore, the turnover rate of ACh in total brain was slower in muscular dystrophic mice than in phenotypically normal litter mates. The turnover rate of brain dopamine and norepinephrine in these 2 groups of animals was similar.     

Pulmonary blood flow and ventilation-perfusion heterogeneity

We studied the independent influence of changes in perfusion on pulmonary gas exchange in the left lower lobe (LLL) of anesthetized dogs. Blood flow to the LLL (QLLL) was raised 50% (increased QLLL) or reduced 50% (decreased QLLL) from baseline by partial occlusion of the right or left pulmonary artery, respectively. Minute ventilation and alveolar PCO2 of the LLL remained constant throughout the study. We determined ventilation-perfusion distributions of the LLL using the multiple inert gas elimination technique. Increased QLLL impaired LLL pulmonary gas exchange. All dispersion indexes and all arterial-alveolar difference areas increased (P less than 0.01). Decreased QLLL increased the log standard deviation of the perfusion distribution (P less than 0.05) and reduced the log standard deviation of the ventilation distribution (P less than 0.01) but did not affect the dispersion indexes or alveolar-arterial difference areas. We conclude that ventilation-perfusion heterogeneity is increased by independent changes in perfusion from normal baseline blood flow, even when ventilation and alveolar gas composition remain constant.