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.     

Chymotrypsin inhibitor from potatoes: interaction with target enzymes

The interactions of chymotrypsin, subtilisin and trypsin with a low MW proteinase inhibitor from potatoes were investigated. The K_i value calculated for the binding of inhibitor to chymotrypsin was 1.6 ± 0.9 × 10^?10M, while the second-order rate constant for association was 6 × 10⁵ M^?1/sec. Although binding was not observed to chymotrypsin which had been treated with diisopropyl fluorophosphate or with l-tosylamide-2-phenylethyl chloromethyl ketone, the 3-methylhistidine-57 derivative bound inhibitor with a K_i value of 9.6 × 10^?9 M. The inhibitor also exhibited a tight association with subtilisin (K_i < 4 × 10^?9 M). In contrast, little inhibition of trypsin was observed, and this was believed to be due to low levels of a contaminant in our preparations. No evidence for reactive site cleavage was observed after incubation of the inhibitor with catalytic amounts of chymotrypsin or subtilisin at acid pH.     

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.     

Processing and fermentation of Jerusalem artichoke for ethanol production

Summary Processing and fermentation trials on Jerusalem artichoke (Helianthus tuberosus L.) tubers, and on pure inulin media were carried out. Acid and thermal treatments, pure and mixed cultures of yeast, and enzyme preparations were investigated. Best ethanol yields on either substrate were obtained with pH 2 thermal treatments, resulting in 131.6 liters ethanol per metric ton fresh tuber.     

The relationship between the size of mitochondria and the intensity of light that they scatter in different energetic states

The intensity of light scattered at 90° to the incident beam and the effective hydrodynamic radii of mitochondria incubated under a variety of conditions have been measured. Addition of high concentrations of uncouplers to respiring mitochondria resulted in a decrease in scatter which was not due to swelling. Addition of valinomycin to mitochondria depleted of substrate in K⁺-free medium produced an increase in scatter that was not due to shrinking. It is concluded that changes in the intensity of scattered light are not reliable indices of changes of volume of mitochondria, and that changes in conformation with changes in metabolic state dominate changes in light scatter. A molecular mechanism for the effect of metabolic state upon the scattered intensity is suggested.     

Tyrosine aminotransferase deficiency in mink (Mustela vision): A model for human tyrosinemia II

Mink pseudodistemper, a recessive disease associated with high blood tyrosinelevels, is an animal analogue of the human inborn error of metabolism, tyrosinemia II. Affected mink and man have eye and skin lesions. Affected mink have no hepatic tyrosine aminotransferase (TAT) activity, as measured immunologically and biochemically. Hepatic mitochondrial aspartate aminotransferase is increased to 188% of control. This new genetic animal model of TAT deficiency should allow new studies of tyrosine metabolism.Supported by NIH Grants AM-17253 (LAG), 5T32-AM-07093 (LAG), and RCDA AM-0008 (LAG), grants from the Wisconsin State Mink Advisory Board, and a BRSG grant for the Graduate School of the University of Wisconsin, Madison, Publication No. 82 of the dermatology research laboratories of Duke University Medical Center.     

Five-year perspective of the large-scale growth of mammalian cells in suspension culture

The Cell Culture Center at the University of Alabama in Birmingham was set up to produce large quantities of cells and their products from suspension cultured cell lines. This system has now been in operation for over five years and has been effective in producing large quantities of mammalian cells of murine and human origin. This article describes the system and some growth parameters which have been of importance for large-scale mammalian cell growth.