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.     

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.     

An evaluation of the phylogenetic position of the dinoflagellateCrypthecodinium cohnii based on 5S rRNA characterization

Summary Partial nucleotide sequences for the 5S and 5.8S rRNAs from the dinoflagellateCrypthecodinium cohnii have been determined, using a rapid chemical sequencing method, for the purpose of studying dinoflagellate phylogeny. The 5S RNA sequence shows the most homology (75%) with the 5S sequences of higher animals and the least homology (< 60%) with prokaryotic sequences. In addition, it lacks certain residues which are highly conserved in prokaryotic molecules but are generally missing in eukaryotes. These findings suggest a distant relationship between dinoflagellates and the prokaryotes. Using two different sequence alignments and several different methods for selecting an optimum phylogenetic tree for a collection of 5S sequences including higher plants and animals, fungi, and bacteria in addition to theC. cohnii sequence, the dinoflagellate lineage was joined to the tree at the point of the plant-animal divergence, well above the branching point of the fungi. This result is of interest because it implies that the well-documented absence in dinoflagellates of histones and the typical nucleosomal subunit structure of eukaryotic chromatin is the result of secondary loss. and not anindication of an extremely primitive state, as was previously suggested. Computer simulations of 5S RNA evolution have been carried out in order to demonstrate that the above-mentioned phylogenetic placement is not likely to be the result of random sequence convergence.We have also constructed a phylogeny for 5.8S RNA sequences in which plants, animals, fungi and the dinoflagellates are again represented. While the order of branching on this tree is the same as in the 5S tree for the organisms represented, because it lacks prokaryotes, the 5.8S tree cannot be considered a strong independent confirmation of the 5S result. Moreover, 5.8S RNA appears to have experienced very different rates of evolution in different lineages indicating that it may not be the best indicator of evolutionary relationships.We have also considered the existing biological data regarding dinoflagellate evolution in relation to our molecular phylogenetic evidence.     

Production of extrachromosomal r-determinant circles from integrated R100.1: Involvement of the E. coli recombination system

Summary The drug resistance plasmid R100.1 can integrate into the E. coli chromosome at several sites on the plasmid. Many of the resulting Hfr strains continuously produce extrachromosomal circular forms of the r-determinant. These r-det plasmids seem incapable of stable autonomous replication. We show that their presence in the cell requires the continuous activity of functional recA and recC genes but does not require the lexA function.The production of r-det circular forms is correlated with an increased copy number of r-det sequences, relative to RTF sequences. This copy number increase is, however, also found in a recA ^- backgound where no circular forms of r-det are found. These results show that a specific replication of r-det sequences, not present in the wild-type R100.1 plasmid, occurs in these R-Hfr strains. They suggest that a rec promoted recombination, posterior to the specific replication event, is needed for the production of circular r-det forms.     

Retention of human skin fibroblast fatty acid modifications during maintenance culture

Summary The fatty acid composition of cultured human skin fibroblasts was modified by adding either oleic or linoleic acid to the growth medium. After the cultures became confluent, they were washed and transferred to different maintenance media in order to determine the stability of the various fatty acyl modifications. Some changes in fatty acid composition occurred under all conditions. When the maintenance medium was supplemented with fatty acid, the cellular neutral lipid and phospholipid fatty acyl composition were altered markedly within 16 to 24 hr. If no supplemental fatty acid was available during the maintenance period, however, the modified fatty acyl compositions were sufficiently retained so that appreciable differences between the cells enriched with oleate and linoleate persisted for at least 48 to 72 hr. This considerable degree of stability occurred when either 10% delipidized fetal bovine serum or 10% fetal bovine serum containing its inherent lipids were present in the maintenance medium. Although the triglyceride content of the fatty acid-modified cells was quite labile, neither the cholesterol nor phospholipid content changed appreciably during culture in any of the maintenance media. Since the fatty acid compositional differences persisted during several days of maintenance under certain conditions, these modified cultures appear to be a useful experimental system for assessing the effect of lipid structure on fairly long-term cellular functions. This work was supported by Arteriosclerosis Specialized Center of Research Grant HL14230 from the National Heart, Lung and Blood Institute, National Institutes of Health.