The use of clip cages to restrain insects reduces leaf expansion systemically in Rumex obtusifolius

Abstract. 1. Clip cages have been used widely by experimental ecologists to contain insects on plants.
2. Under controlled conditions, the effect of applying clip cages alone and clip cages and the chrysomelid beetle Gastrophysa viridula on systemic leaf expansion of Rumex obtusifolius was investigated. Treatments were applied to the fully expanded fourth leaf and expansion of leaf 8 was measured over a period of 22 days.
3. The application of clip cages reduced the rate at which leaf area increased and led to reductions in final leaf areas.
4. Clip cages have systemic effects on plant development and these effects are sustained even after the clip cage is removed. Investigators should take this into account in designing experiments.     

Proteolytic processing of foot-and-mouth disease virus polyproteins expressed in a cell-free system from clone-derived transcripts

All picornaviral genes are expressed as a single, large polyprotein, which is proteolytically processed into the system produces functional proteins, including viral protease 3C, which plays a major role in processing the precursor proteins. To study the function of the two putative proteases 3C and leader (L) in processing, we constructed several cDNA plasmids encoding various regions of the FMDV type A12 genome. These plasmids, containing FMDV cDNA segments under the control of the T7 promoter, were transcribed in vitro by using T7 RNA polymerase and then translated in rabbit reticulocyte lysates. The expressed FMDV gene products were identified by immunoprecipitation with specific antisera and analyzed by gel electrophoresis. The results demonstrate the following: (i) the leader protein, L, is processed from the structural protein precursor, P1, in the absence of any P2 or P3 region proteins; (ii) protein 2A remains associated with the structural protein precursor, P1, rather than the precursor, P2; (iii) the processing of the P1-2A/P2 junction is not catalyzed by 3C or L; (iv) the proteolytic processing of polyproteins from the structural P1 region (except VP4/VP2) and the nonstructural P2 and P3 region is catalyzed by 3C.     

Purification and Properties of Rubrophilin: A Novel Brain Specific Membrane Polypeptide

Rubrophilin, a unique brain specific polypeptide, was purified to apparent homogeneity from microsomal fractions of bovine brains. The peptide stains pink with Coomassie Brilliant Blue R-250 (C.I. No. 42660) under specific conditions, has an apparent Mr of 53,000, and is acidic with an apparent pI of 4.9. The purification involves initial solubilization of delipidated microsomes in sodium dodecyl sulfate, followed by ammonium sulfate fractionation, reversed ammonium sulfate gradient elution from diatomaceous earth, gel filtration on polyacrylamide (Biogel P-200), gradient elution chromatography from hydroxylapatite, and reverse-phase chromatography from phenyl-Sepharose. A yield of about 5 mg of rubrophilin was obtained from 9 g of microsomal proteins. Amino acid analysis shows that rubrophilin contains only nine amino acids with residues/mol as follows: alanine (102), glutamic acid (97), lysine (65), proline (55), aspartic acid (48), glycine (44), serine (37), threonine (35), and valine (10). Cysteine, methionine, tryptophan, tyrosine, isoleucine, phenylalanine, histidine, and arginine could not be detected. Relative rubrophilin content of vertebrate brains was as follows: mammals greater than birds greater than reptiles greater than fishes. It is present in mouse retina and human neuroblastoma cell cultures but could not be detected in octopus optic lobe or in cultured C-6 rat glioma cells.     

Influence of microgravity on cellular differentiation in root caps of Zea mays

We launched imbibed seeds of Zea mays into outer space aboard the space shuttle Columbia to determine the influence of microgravity on cellular differentiation in root caps. The influence of microgravity varied with different stages of cellular differentiation. Overall, microgravity tended to 1) increase relative volumes of hyaloplasm and lipid bodies, 2) decrease the relative volumes of plastids, mitochondria, dictyosomes, and the vacuome, and 3) exert no influence on the relative volume of nuclei in cells comprising the root cap. The reduced allocation of dictyosomal volume in peripheral cells of flight-grown seedlings correlated positively with their secretion of significantly less mucilage than peripheral cells of Earth-grown seedlings. These results indicate that 1) microgravity alters the patterns of cellular differentiation and structures of all cell types comprising the root cap, and 2) the influence of microgravity on cellular differentiation in root caps of Zea mays is organelle specific.     

Cricket Paralysis Virus, a Potential Control Agent for the Olive Fruit Fly, Dacus oleae Gmel

Representatives of several families of insect viruses were tested for growth and pathogenicity in the olive fruit fly, Dacus oleae Gmel. The viruses included nuclear polyhedrosis viruses, an iridovirus, two picornaviruses, and Trichoplusia ni small RNA virus (a member of the Nudaurelia β family), in addition to two naturally occurring viruses of the olive fruit fly. Two viruses, one of the two picornaviruses (cricket paralysis virus [CrPV] and the iridovirus (type 21 from Heliothis armigera), were found to replicate in adult flies. Flies which were fed on a solution containing CrPV for 1 day demonstrated a high mortality with 50% dying within 5 days and nearly 80% dying within 12 days of being fed. The virus was transmissible from infected to noninfected flies by fecal contamination. The CrPV which replicated in the infected flies was demonstrated to be the same as input virus by infection of Drosophila melanogaster cells and examination of the expressed viral proteins, immunoprecipitation of the virus purified from flies, and electrophoretic analysis of the structural proteins.     

Isolation of Different Agrobacterium Biovars from a Natural Oak Savanna and Tallgrass Prairie

Populations of agrobacteria in excess of 10⁵ CFU/g were recovered from 12 soil and root samples obtained from the Allison Savanna, Minn., a natural oak savanna and tallgrass prairie which has never been disturbed agriculturally. Of 126 strains picked randomly from selective media, 54 were identified as Agrobacterium spp. Biovar 2 strains predominated (35 of 54), but these strains were distributed into three phenotypically distinct subgroups. Of the remaining Agrobacterium strains, four were biovar 1-2, one was biovar 1, and none were biovar 3. The last 14 Agrobacterium strains formed a homogeneous group which differed biochemically from the hitherto reported biovars. Opine utilization (coded for by genes on the tumor-inducing plasmid in pathogenic Agrobacterium spp.) by these agrobacteria was limited to two biovar 2 strains. In contrast, 10 nonfluorescent gram-negative strains utilized either nopaline or octopine as the sole carbon and nitrogen source. There may be a need to reexamine the source and role of opines in the terrestrial environment because (i) all of these opine utilizers were isolated from an environment free of crown gall, the only known terrestrial source of opines, and (ii) 83% of the opine utilizers were not Agrobacterium spp.     

Complementary Methodologies To Identify Specific Agrobacterium Strains

Serological techniques and restriction enzyme cleavage patterns of total DNA were used to differentiate strains of Agrobacterium spp. Forty-five wild-type and plasmid-cured Agrobacterium strains were tested by immunodiffusion and immunofluorescence against polyclonal antisera to a crude ribosome preparation from Agrobacterium strains K84, U11, B6, A323, NT1, and C58. In immunodiffusion gels, these antisera reacted only with water-phenol extracts of the homologous strain, producing a single, strain-specific precipitin line. In contrast, when the same antisera were used in immunofluorescence staining, cross-reactions occurred with a limited number of heterologous Agrobacterium strains. However, the cross-reacting heterologous cells fluoresced generally less brightly than the homologous cells. When the EcoRI-digested DNA profiles from the same Agrobacterium strains were compared, 34 distinct cleavage patterns were observed. The DNA profiles were the same for all strains sharing a common chromosomal background and correlated with the strain-specific serological reaction. The presence or absence of plasmid DNA did not alter the strain-specific serological reaction or the DNA cleavage patterns. Both the serological reaction and the restriction enzyme digestion of total DNA were complementary to each other. These methods were used successfully to identify A. radiobacter K84 strains which were recovered 6 months after being inoculated to young trees in the field.     

Abnormal subcellular distribution of β-glucuronidase in mice with a genetic alteration in enzyme structure

Liver -glucuronidase is structurally altered in inbred strain PAC so that a peptide subunit with a more basic isoelectric point, GUS-SN, is produced. This allele of -glucuronidase was transferred to strain C57BL/6J by 12 backcross matings to form the congenic line B6 · PAC-Gus ⁿ. Liver -glucuronidase activity was halved in males of the congenic strain compared to normal males. The lowered activity was specifically accounted for by a decrease in the lysosomal component. There was no alteration in the concentration of microsomal activity. This alteration in the subcellular distribution of -glucuronidase in Gus ⁿ/Gus ⁿ mice was confirmed by two independent gel electrophoretic systems which separate microsomal and lysosomal components. -Glucuronidase activity was likewise approximately halved in mutant spleen, lung, and brain, organs which contain exclusively or predominantly lysosomal -glucuronidase. The loss of liver lysosomal -glucuronidase activity was shown by immunotitration to be due to a decrease in the number of -glucuronidase molecules in lysosomes of the congenic strain. The Gus ⁿ structural alteration likely causes the lowered lysosomal -glucuronidase activity since the two traits remain in congenic animals. Heterozygous Gus ⁿ/Gus ^b animals had intermediate levels of liver -glucuronidase. Also, the effect was specific, in that three other lysosomal enzymes were not reproducibly lower in Gus ⁿ/Gus ⁿ mice. Gus ⁿ is, therefore, an unusual example of a mutation which causes a change in the subcellular distribution of a two-site enzyme.This work was supported by National Institutes of Health Grants GM-33559 and GM-33160 and National Science Foundation Grant PCM-8215808.     

Secondary structure of 5S RNA: NMR experiments on RNA molecules partially labeled with nitrogen-15

A method has been found for reassembling fragment 1 of Escherichia coli 5S RNA from mixtures containing strand III (bases 69-87) and the complex consisting of strand II (bases 89-120) and strand IV (bases 1-11). The reassembled molecule is identical with unreconstituted fragment 1. With this technique, fragment 1 molecules have been constructed 15N-labeled either in strand III or in the strand II-strand IV complex. Spectroscopic data obtained with these partially labeled molecules show that the terminal helix of 5S RNA includes the GU and GC base pairs at positions 9 and 10 which the standard model for 5S secondary structure predicts [see Delihas, N., Anderson, J., & Singhal, R. P. (1984) Prog. Nucleic Acid Res. Mol. Biol. 31, 161-190] but that these base pairs are unstable both in the fragment and in native 5S RNA. The data also assign three resonances to the helix V region of the molecule (bases 70-77 and 99-106). None of these resonances has a "normal" chemical shift even though two of them correspond to AU or GU base pairs in the standard model. The implications of these findings for our understanding of the structure of 5S RNA and its complex with ribosomal protein L25 are discussed.     

Isolation and culture of cells derived from human cerebral microvessels

Summary Microvessels were isolated from non-neoplastic human cerebral cortical fragments resected for treatment of intractable seizure disorder. The microvessels were incubated in modified Lewis medium with 20 or 30% fetal bovine serum. Within 1–2 weeks, two cell populations emerged from the isolates. One type of cells had polygonal morphology, showed density-dependent contact inhibition at confluence in vitro, showed lectin-binding characteristics of endothelium (but only moderate positivity for factor VIII antigen), demonstrated induction of -glutamyl trans-peptidase when exposed to astrocyte-conditioned media, and responded to insulin by a pronounced increase in DNA synthesis. The other variety of cells grew in vitro more slowly in irregular strands separated by clear zones, showed ultrastructural features of smooth muscle, and isoelectric focusing of cell proteins revealed the presence of smooth-musclespecific -isoactin. Both types of cells could be serially subcultured. The ability to isolate and grow the two cell types, tentatively identified as human cerebral microvascular endothelium and smooth muscle, may facilitate studies of human blood-brain barrier function as well as the pathogenesis of cerebral microangiopathies unique to the human brain.Funded by Canadian Heart Foundation, Heart and Stroke Foundation of Ontario and UCLA Biomedical Research Support Grant