Phylogenetic relationships within the class Oligohymenophorea,phylum Cilophora,inferred from the complete small subunit rRNA gene sequences ofColpidium campylum,Glaucoma chattoni,andOpisthonecta henneguyi

Summary Phylogenetic relationships within the class Oligohymenophorea, phylum Ciliophora, were investigated by determining the complete small subunit rRNA (SSrRNA) gene sequences for the hymenostomesColpidium campylum, Glaucoma chattoni, and the peritrichOpisthonecta henneguyi. The affiliations of the oligohymenophoreans were assessed using both distance matrix (DM) and maximum parsimony (MP) analyses. Variations do exist in the phylogenies created by the two methods. However, the basic tree topologies are consistent. In both the DM and MP analyses the hymenostomes (C. campylum, G. chattoni, and the tetrahymenas) all form a very tight group associated with the peritrichO. henneguyi. TheTetrahymena lineage was monophyletic whereasColpidium andGlaucoma were more closely related to each other than either was to the tetrahymenas. The monophyly of the genusTetrahymena in the present analysis supports the phylogenies determined from morphological data and molecular sequence data from the histone H3II/H4II region of the genome. The perplexing and controversial phylogenetic position of the peritrichs is once again depicted in the present analysis. The distinctiveness of the peritrichOpisthonecta from both hymenostome and nassophorean ciliates based on evolutionary distances suggests that the elevation of the peritrichs to a higher taxonomic rank should be reconsidered.     

Chusquea sect. Swallenochloa (Poaceae: Bambusoideae) and allies in Brazil

The 13 high altitude/latitude, dwarf species ofChusquea in Brazil are described, illustrated, and mapped, and their morphology, habitats, distributions, and taxonomic affinities are discussed. Two keys to species are provided, one based solely on vegetative characters, and the other on vegetative and flowering characters.Chusquea erecta, C. nutans, C. riosaltensis,C. windischii, C. caparaoensis, andC. nudiramea are described as new, andC. microphylla is elevated to specific status. Two subspecies are recognized within the variableC. mimosa: C. mimosa subsp.australis and subsp.mimosa. Seven species are formally classified withinChusquea sect.Swallenochloa; the remaining six species are classified into two informal categories, theNudiramea andHeterophylla groups. A list of all the species currently included withinChusquea sect.Swallenochloa is provided.     

Probing nucleosome core secondary structure before and after α-chymotrypsin treatment by raman spectroscopy and thermal denaturation

Nucleosome cores were digested with α-chymotrypsin until histone H3 was degraded to a partial histone, CP1. As we reported previously, cleavage occurred at leucine 20 to H3 and resulted in an increase in circular dichroism between 265 to 285 nm. Some modest core unfolding was also observed as determined by a small decrease in the sedimentation coefficient. Studies reported here deal with the analysis of core secondary structure and subsequent perturbation caused by treatment with α-chymotrypsin. Raman spectroscopy indicated that chymotryptic treatment promoted a change in the conformational environment of a population of core histone tyrosines. In addition, a shift from B-form to an intermediate B- or A-form was observed for core DNA. High-resolution thermal denaturation was used to determine alterations in the stabilization of core DNA related to perturbation of the core histones. Brief chymotryptic treatment indicated changes in both pre-melt and irreversible transitions.     

Aminopyridines block an inactivating potassium current having slow recovery kinetics.

The blocking action of aminopyridines on an inactivating K current (lKi) in GH3 pituitary cells was studied before and after altering the macroscopic decay of the current with N-bromoacetamide (NBA). The first depolarizing pulse delivered either seconds or minutes after beginning 4-aminopyridine (4AP) application, elicited a current with both a more rapid decay and a reduced peak amplitude. The rapid decay (or time-dependent block) was especially prominent in NBA-treated cells. With continued drug application, subsequent test pulses revealed a stable block of peak current, greater in NBA-treated than control cells. Recovery from block was enhanced by hyperpolarizing holding potentials and by the first depolarizing pulse delivered after prolonged recovery intervals. Unlike aminopyridine block of other K currents, there was no convincing evidence for voltage shifts in activation or inactivation, or for voltage and frequency-dependent unblock. Increasing the open probability of the channels did, however, facilitate the block. Although the behavior of currents in 4AP was suggestive of "open channel block," the block was not produced by 4-aminopyridine methiodide, a positively charged aminopyridine. Moreover, because partial block and recovery occurred without opening the channels we suggest that aminopyridines bind to, or near, this K channel, that this binding is enhanced by opening the channel, and that a conformational change is induced which mimics inactivation. Because recovery from block is enhanced by negative potentials, we suggest that aminopyridine molecules may become "trapped" by inactivation awaiting the slow process of reactivation to escape their binding sites.     

Biological Control of Damping-Off of Alfalfa Seedlings with Bacillus cereus UW85

We explored the potential of biological control of alfalfa (Medicago sativa L.) seedling damping-off caused by Phytophthora megasperma f. sp. medicaginis by screening root-associated bacteria for disease suppression activity in a laboratory bioassay. A total of 700 bacterial strains were isolated from the roots of field-grown alfalfa plants by using Trypticase soy agar. A simple, rapid assay was developed to screen the bacteria for the ability to reduce the mortality of Iroquois alfalfa seedlings that were inoculated with P. megasperma f. sp. medicaginis zoospores. Two-day-old seedlings were planted in culture tubes containing moist vermiculite, and each tube was inoculated with a different bacterial culture. Sufficient P. megasperma f. sp. medicaginis zoospores were added to each tube to result in 100% mortality of control seedlings. Of the 700 bacterial isolates tested, only 1, which was identified as Bacillus cereus and designated UW85, reduced seedling mortality to 0% in the initial screen and in two secondary screens. Both fully sporulated cultures containing predominantly released spores and sterile filtrates of these cultures of UW85 were effective in protecting seedlings from damping-off; filtrates of cultures containing predominantly vegetative cells or endospores inside the parent cell had low biocontrol activity. Cultures grown in two semidefined media had significantly greater biocontrol activities than cultures grown in the complex tryptic soy medium. In a small-scale trial in a field infested with P. megasperma f. sp. medicaginis, coating seeds with UW85 significantly increased the emergence of alfalfa. The results suggest that UW85 may have potential as a biocontrol agent for alfalfa damping-off, thus providing an alternative to current disease control strategies.     

Nonshivering thermogenesis and cold resistance during seasonal acclimatization in the Djungarian hamster

Summary The absence of juvenile hormone (JH) at the time of head capsule slippage during the molt to the fifth (final) instar of the tobacco hornworm was found to cause ommochrome (primarily dihydroxanthommatin) synthesis in the epidermis during the first two days after ecdysis. Then synthesis decreased until its transient reappearance during the wandering stage. Either JH-I (ED₅₀=8x10^–4 g) or methoprene (ED₅₀=1.4x10^–2 g) applied at this critical time during the molt prevented the first synthesis. A comparison of developmental profiles of tryptophan and its metabolites, kynurenine and 3-hydroxykynurenine, in normal and allatectomized wild type larvae showed that JH at this critical time prevented both the conversion of kynurenine to 3-hydroxykynurenine and 3-hydroxykynurenine to ommochromes. A similar study in normal and methoprene-treatedblack mutant larvae showed that only the latter conversion was inhibited by JH. The accumulation of 3-hydroxykynurenine in the epidermis of the JH-treatedblack mutant is thought to be due to the altered tryptophan metabolism in these mutants in previous instars due to lower JH levels. Neither starvation of theblack mutant nor injection of 3-hydroxykynurenine significantly affected ommochrome synthesis by the epidermis. Preliminary studies of the enzymes involved showed that JH at the critical period suppressed the later activity and/or production of kynurenine 3-hydroxylase in the wild type larva, but had little effect on the particulate ommochrome synthetase activity of the epidermis.Abbreviations CA corpora allata - JH juvenile hormone - PTTH prothoracicotropic hormone     

Identification of surface autoantigens which appear during spermatogenesis

Autoantigens that appear during spermatogenesis in the rabbit were identified using immunoadsorbent chromatography and SDS-PAGE. To identify cell-surface proteins, samples of freshly isolated, staged cells were labeled by the lactoperoxidase or Iodo-Gen iodination procedure and run on SDS-PAGE. Autoradiograms of the stained, dried gels were prepared. By correlating the band patterns in the SDS gels of immunocolumn and surface-labeled samples with the band patterns in the autoradiograms, it was possible to show when the autoantigenic proteins appeared on the cell surface. To further support the identification of membrane autoantigens, surface-labeled, staged cell samples were lysed in Triton X-100 and immunoprecipitated with antitestis cell autoantisera. Three types of autoantigens have been identified: (1) late class antigens that are present only on late spermatids and epididymal spermatozoa, but are intracellular in early stages, (2) early class antigens which occur on the surface of pachytene spermatocytes and are present throughout subsequent stages of development, and (3) early class, transient antigens, which appear on spermatogenic cells but are not present on epididymal spermatozoa.     

Effects of methoprene and juvenile hormone on larval ecdysis,emergence, and metamorphosis of the endoparasitic wasp, Apanteles congregatus

Topical application of juvenile hormone I and III or the hormone analogue methoprene to parasitized Manduca sexta larvae inhibited subsequent emergence of the endoparasitic wasp Apanteles congregatus. Methoprene treatment inhibited wasp emergence in a dose-dependent manner, causing either a delay or total inhibition of emergence. These results were interpreted as reflecting inhibitory effects of juvenile hormone on the second-larval ecdysis of the parasitoid that normally occurs during emergence from the host larva. Parasitoid ecdysis was disrupted even when methoprene was applied to host larvae a few hours prior to the normal expected time of emergence. A correlation between the number of emerging parasitoids and the timing of emergence was seen in methoprene-treated hosts, and few parasitoids emerged after day 9 of the host's fifth-instar. Our findings suggest that the suppression of emergence by juvenile hormone analogues noted in previous studies may be due to a similar inhibitory effect on parasitoid ecdysis. We also observed that parasitoids emerging from hosts treated with a low dose of methoprene (1 μg) later pupated normally but then formed nonviable pupal-adult intermediates. Thus use of this insect growth regulator must be undertaken carefully to prevent possible adverse effects on natural parasitoid populations.     

Complete amino acid sequence of a variant surface glycoprotein (VSG 117) from Trypanosoma brucei

The amino acid sequence of a variant surface glycoprotein (VSG 117) of Trypanosoma brucei has been determined by manual sequencing of tryptic. staphylococcal protease and cyanogen bromide peptides and fragments derived from these peptides. Some overlaps needed for completion of the sequence were deduced from the nucleotide sequence of complementary DNA derived from messenger RNA coding for VSG 117. The glycoprotein consists of 470 amino acid residues with two carbohydrate chains attached at Asn420 and Asp470. No pronounced hydrophobic regions, which are characteristic of many membrane proteins, are present in the isolated glycoprotein, and the carboxy-terminal region, which is close to the membrane, is remarkably hydrophilic. These observations indicate that the molecule probably does not penetrate the lipid bilayer of the plasma membrane. The high proportion of charged residues in the carboxyterminal region is more consistent with electrostatic interaction with the polar head groups of the phospholipids.     

Some kinetic and steady-state properties of sodium channels after removal of inactivation

To study the kinetic and steady-state properties of voltage-dependent sodium conductance activation, squid giant axons were perfused internally with either pronase or N-bromoacetamide and voltage clamped. Parameters of activation, tau m and gNa(V), and deactivation, tau Na, were measured and compared with those obtained from control axons under the assumption that gNa oc m3h of the Hodgkin-Huxley scheme. tau m(V) values obtained from the turn-on of INa agree well with control axons and previous determinations by others. tau Na(V) values derived from Na tail currents were also unchanged by pronase treatment and matched fairly well previously published values. tau m(V) obtained from 3 x tau Na(V) were much larger than tau m(V) obtained from INa turn-on at the same potentials, resulting in a discontinuous distribution. Steady-state In (gNa/gNa max - gNa) vs. voltage was not linear and had a limiting logarithmic slope of 5.3 mV/e-fold gNa. Voltage step procedures that induce a second turn-on of INa during various stages of the deactivation (Na tail current) process reveal quasiexponential activation at early stages that becomes increasingly sigmoid as deactivation progresses. For moderate depolarizations, primary and secondary activation kinetics are superimposable. These data suggest that, although m3 can describe the shape of INa turn-on, it cannot quantitatively account for the kinetics of gNa after repolarization. Kinetic schemes for gNa in which substantial deactivation occurs by a unique pathway between conducting and resting states are shown to be unlikely. It appears that the rate-limiting step in linear kinetic models of activation may be between a terminal conducting state and the adjacent nonconducting intermediate.