Isolated hepatocytes fixed on collagen, an accurate model for the the secretion of proteins in a minimal medium. Response to inflammation

The albumin, orosomuco?d and alpha 2-macroglobulin secretion by isolated hepatocytes of normal and suffering from Turpentine-induced inflammation rats, is investigated for 4 hr. The model, stable over the whole duration of incubation, is a true reflect of hepatic secretion in vivo and can be used to measure it.     

Efficient lipid-mediated transfection of DNA into primary rat hepatocytes

Cationic lipids are an effective means for transfecting nucleic acids into a variety of cell types. Very few of these lipids, however, have been reported to be effective with primary cells. We report on the efficacy of several commercially available cationic lipid reagents to transfect plasmid DNA into primary rat hepatocytes in culture. The reagents tested in this study include TransfectAce, LipofectAmine, Lipofectin, N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammoniumchloride (DOTMA), (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methylsulfate (DOTAP), and cetyltrimethyl-ammonium bromide/dioleoylphosphatidylethanol-amine (CTAB/DOPE). Electron micrographic (EM) studies indicate that similar size Lipofectin and DOTAP vesicles contain DNA-like material internally and that these vesicles attach to the cell membrane. DOTAP vesicles are multilamellar, appear as clusters, and have a high DNA-to-lipid ratio. Lipofectin vesicles appear to attach to the cell surface as individual vesicles. The EM observations are consistent with current theories on the mechanism of transfection by cationic lipids. While Lipofectin has proven to be effective in transfection studies of primary cells in culture, we have found DOTAP to be a viable alternative. DOTAP yields transfection rates in hepatocytes comparable to DOTMA and Lipofectin, however, at lower concentrations of reagent and at considerably less cost. Optimal conditions for transfecting 5 μg of plasmid DNA with DOTAP were achieved by utilizing multilamellar (vortexed) vesicles at a concentration of 15 μg DOTAP per 2 ml media in 60-mm plates for 2 h transfection time. In this study, DOTAP has proven to be economical, easy to prepare, and very effective in transfecting DNA into primary rat hepatocytes.     

Expression of a parasitism-specific protein in lepidopteran hosts of Chelonus sp.

The hemolymph of each noctuid species successfully parasitized by Chelonus near curvimaculatus possessed a parasitism-specific protein (PSP) previously identified in host T. ni (Insect Biochem. 19:445; 21:845). Expression of PSP occurred in a stage-specific manner in the stadium during which the host undergoes precocious metamorphosis. The appearance of the protein was not due to nutritional stress associated with parasitism of hosts, since starved nonparasitized larvae did not produce the protein, or to low juvenile hormone titers occurring in precociously metamorphosing hosts, but rather was dependent on the presence of the endoparasite larva. Results of in vivo incorporation experiments with [³⁵S]-methionine showed that synthesis and subsequent appearance of the protein in the hemolymph of parasitized hosts was abrogated by prior surgical removal of endoparasite. Immunoprecipitation analysis of proteins from C. near curvimaculatus larvae cultured in vitro using antibodies specific to PSP indicated that the source of the protein was the endoparasite. Synthesis of PSP by the endoparasitic larvae with its subsequent secretion into the hemocoel of hosts was specific to the advanced stages of parasite development prior to its egression from the host. © 1993 Wiley-Liss, Inc.     

Genetic transfer of clumping phenotype inAnacystis nidulans

Occasionally a mutation occurs in liquid cultures ofAnacystis nidulans, spreading quickly through the population and causing cells to adhere together in clumps. This phenotype is stable indefinitely and is an inherited characteristic of all cells within a clumping culture. Inoculation with a few living cells from a clumping culture quickly produces the clumping genotype in a majority of cells within a previously non-clumping culture. Killed cells, broken cell extracts, or media from clumping cultures do not produce aggregation in non-clumping cultures. Actively growing cells in clumping cultures do not affect non-clumping cultures when separated by 0.4 μm Millipore filter. Apparently transfer of the clumping trait requires direct contact between living cells. Pili-like projections connect individual cells within clumps, but no slime layer or capsule is seen. Clumps can be dispersed without cell damage; reaggregation requires photosynthesis.     

Juvenile hormone esterases of Lepidoptera

Summary The juvenile hormone esterase (JHE) titer was measured during the last larval instar of 11 species of Lepidoptera (Pieris rapae, Junonia coenia, Danaus plexippus, Hemileuca nevadensis, Pectinophora gossypiella, Spodoptera exigua, Orgyia vetusta, Ephestia elutella, Galleria mellonella, Manduca sexta andEstigmene acrea). All species had a peak of JHE at or near the time of wandering. The peak activity at this time ranged from 0.8 to 388 nmoles JH III cleaved/min·ml. All species exceptJ. coenia had a second peak of JHE during the late prepupal stage. The height of the second peak ranged from 0.4 to 98.4 nmoles/min·ml. However, there was no apparent correlation between size of the first and second JHE activity peaks for the lepidopteran species examined. There was an apparent relationship between the height of the first and second JHE peaks and reports on titer of JH just prior to these peaks. These data support, with some qualifications, the extension of developmental information obtained on several well studied species to a variety of Lepidoptera.Abbreviations JH juvenile hormone - JHE juvenile hormone esierase - PTTH prothoracotropic hormone - R _o -10-3108 1-(4-ethylphenoxy)-6,7-epoxy-3-ethyl-7-methylnonane     

Predators and parasites of temporary row crop pests: Agents of irreplaceable mortality or scavengers acting prior to other mortality factors?

A study was conducted with the cabbage looper,Trichoplusia ni (Hübner), to determine: 1) whether naturally occurring entomophagous arthropods impart irreplaceable mortality to the cabbage looper in celery, and 2) whether entomophagous arthropods are present to impart such mortality toT. ni eggs and 1st instar larvae when the crop is sensitive to the pest. Predator evaluation involved 1) insecticidal + cage exclusion, 2) insecticidal exclusion alone, 3) D-vac removal of predators, and 4) sticky barrier exclusion. In all the exclusion regimes survival of eggs and larvae was higher than in the unexcluded control. Analysis of life table data was consistent with the hypothesis that some mortality of eggs due to parasitism byTrichogramma and of medium-larvae by other hymenopterous parasites was not replaceable by other mortality factors. The time of appearance of the first marketable petiole was correlated with both the height of the tallest petiole and the number of petioles on the plant. Peak densities ofTrichogramma and important predators occurred before the first marketable petiole appeared and declined to low levels as harvest approached.     

Effects of acute and chronic restraint and estrus cycle on pituitary-adrenal function in the rat

Female Long-Evans hooded rats with 5-day estrus cycles were subjected to 4 hr of continuous restraint for either 1 or 20 days. On the last day of the stress regimen, plasma and adrenal corticosterone concentrations were determined and classified according to the stage of the estrous cycle. The results indicated that acute stress produced greater plasma corticosterone concentrations than controls only during estrus, whereas in response to chronic stress significant stress-induced increments were observed during estrus and proestrus. The results suggest that the estrous cycle influences the magnitude of the stress-induced increments for both acute and chronic stress. In addition, the pituitary-adrenal system did not show habituation to repeated administration of this stress, but sensitization was observed during proestrus.     

Chelonus sp. near curvimaculatus venom proteins: Analysis of their potential role and processing during development of host Trichoplusia ni

The venom that Chelonus sp. near curvimaculatus injects into each parasitized Trichoplusia ni egg is entirely injected within the first 8 s of the 19-s oviposition period, before deposition of the parasitoid egg that is injected during the final 1-2 s of the oviposition. The parasitization factor, causing precocious metamorphosis of the host, is injected after the venom, but before the parasite egg. The venom by itself does not cause developmental redirection of the host. Chelonus venom proteins are very stable in the host egg during the first 2 days of egg development. Then, on the last day before hatching, they are rapidly degraded by the proteolytic enzymes appearing in 3-day-old T. ni eggs. Among those that degrade the venom proteins are serine-type proteinases, and at least one seems to be a trypsin-like enzyme.