Assessment of virus production and chloramphenicol acetyl transferase expression by insect cells in serum-free and serum-supplemented media using a temperature-sensitive baculovirus

Spodoptera frugiperda insect cells were grown in Sf-900 serum-free medium and two kinds of serum-supplemented media (IPL -41 and Grace's). The specific growth rates of uninfected cells were found to be 0.024, 0.35, and 0.034 h(-1) respectively, at 33 degrees C. The IPL -41 medium supported to highest maximum cell density (10.6 x 10(6) cells/mL) compared to 3.5 x 10(6) and 8.7 x 10(6) cells/mL with the Grace's and serum-free media, respectively. In temperature shifdown experiments with a temperature-sensitive baculo-virus (acts10YM1CAT), virus titer and chloramphenicol acetyl transferase (CAT) expression were highest in the IPL -41 (5.1 x 10(7) PFU/mL and 20000 U/mL). Use of Grace's medium gave higher virus titers than the serum-free medium (4.4 x 10(6) vs 4.1 x 10(5) PFU/mL) as well as higher CAT titers (7050 vs 1980 U/mL). Interestingly, in the three media used, the highest virus and CAT titers were obtained at MOI (multiplicity of infection) of 0.02 At MOI of 2.0 virtually no increase in virus of CAT titer was observed. This result is contrary to those obtained at constant-temperature (27 degrees C) infection and cell culture, in which higher virus titers and recombinant protein expression and obtained at higher MOI.     

Chronic electrical stimulation of nongrafted and grafted skeletal muscles in rats

Our purpose was to determine the effects of chronic electrical stimulation on the structure and function of neve-intact grafts in rats. Fourteen days after grafting, extensor digitorum longus (EDL) grafts (n = 6) and nongrafted EDL muscles (n = 4) were stimulated 8 h/day at 10 Hz for 26 days. Measurements were made subsequently of cytochrome c concentration, capillary density, contraction and relaxation times, developed tension, and the resistance to fatigue. Compared with contralateral nonstimulated grafts, chronically stimulated grafts demonstrated a 65% greater cytochrome c concentration, 45% greater number of capillaries per millimeter squared, 30% greater resistance to fatigue, 35% longer contraction time, 30% longer relaxation time, and 30% lower maximum tetanic tension. The differences that resulted from the stimulation of nongrafted EDL muscles were significant but of less magnitude. Chronic stimulation of 8 h/day provided a mixed stimulus for adaptation that enhanced the metabolic and endurance characteristics of fibers in muscles and grafts, but decreased the total fiber cross-sectional area and development of force.     

Conservation planning for freshwater–marine carryover effects on Chinook salmon survival

Experiences of migratory species in one habitat may affect their survival in the next habitat, in what is known as carryover effects. These effects are especially relevant for understanding how freshwater experience affects survival in anadromous fishes. Here, we study the carryover effects of juvenile salmon passage through a hydropower system (Snake and Columbia rivers, northwestern United States). To reduce the direct effect of hydrosystem passage on juveniles, some fishes are transported through the hydrosystem in barges, while the others are allowed to migrate in‐river. Although hydrosystem survival of transported fishes is greater than that of their run‐of‐river counterparts, their relative juvenile‐to‐adult survival (hereafter survival) can be less. We tested for carryover effects using generalized linear mixed effects models of survival with over 1 million tagged Chinook salmon, Oncorhynchus tshawytscha (Walbaum) (Salmonidae), migrating in 1999–2013. Carryover effects were identified with rear‐type (wild vs. hatchery), passage‐type (run‐of‐river vs. transported), and freshwater and marine covariates. Importantly, the Pacific Decadal Oscillation (PDO) index characterizing cool/warm (i.e., productive/nonproductive) ocean phases had a strong influence on the relative survival of rear‐ and passage‐types. Specifically, transportation benefited wild Chinook salmon more in cool PDO years, while hatchery counterparts benefited more in warm PDO years. Transportation was detrimental for wild Chinook salmon migrating early in the season, but beneficial for later season migrants. Hatchery counterparts benefited from transportation throughout the season. Altogether, wild fish could benefit from transportation approximately 2 weeks earlier during cool PDO years, with still a benefit to hatchery counterparts. Furthermore, we found some support for hypotheses related to higher survival with increased river flow, high predation in the estuary and plume areas, and faster migration and development‐related increased survival with temperature. Thus, pre‐ and within‐season information on local‐ and broad‐scale conditions across habitats can be useful for planning and implementing real‐time conservation programs.     

Functional deficits in medial gastrocnemius grafts in rats: relation to muscle metabolism and beta -AR regulation

Larkin, Lisa M., John A. Faulkner, Richard T. Hinkle, CherylA. Hassett, Mark A. Supiano, and Jeffrey B. Halter.Functional deficits in medial gastrocnemius grafts in rats:relation to muscle metabolism and -AR regulation.J. Appl. Physiol. 83(1): 67-73, 1997.This study tested the hypothesis that alterations in the metabolic integrity of grafted muscle contribute to its diminished ability to sustain power. Compared with control muscles, muscles studied 120 days after the grafting procedure had lower specific forceand sustained power. The sustained power protocol resulted in adepletion of muscle glycogen in control (83%) and grafted (85%)animals. Grafts had lower pre- and poststimulation glycogen, diminishedcitrate synthase activity, and greater hexokinase activity. Nodifferences were observed in phosphofructokinase activity, glucosetransporter GLUT-4 content, fiber type, -adrenergic-receptor (-AR) density, or binding affinity. Isoproterenol-stimulated adenylyl cyclase activity was lower in grafted vs. control muscle, suggesting an uncoupling of the -AR-effector complex. Thus the diminished ability of the grafted muscle to sustain power may beexplained, in part, by a decrease in energy available from glycogenstores and/or a decrease in oxidative capacity.

     

Replication patterns and cytopathology of cells infected with baculoviruses

Conclusion In vitro studies have contributed greatly to an understanding of viral cytopathology, molecular biology, and pathogenesis. A model of the role of baculoviruses in a host-parasite relationship is developing which reveals the virus as gaining control of many aspects of host cell biology including control of the cell replication machinery (apoptotic response, macromolecular synthesis), the cytoskeletal structure, the nuclear membrane and intranuclear architecture. Baculovirus replication is a collection of independent but inter-related processes which work within the framework of the host cell, with the in vivo goal of maximizing production of progeny virions. Further molecular dissection of baculovirus replication should yield insight into the processes and principles of viral and host regulatory systems, perhaps facilitating development of new generations of high efficiency sub-viral expression vector systems and the development of genetically improved strains of virus safe for field use in ecologically based pest management strategies.     

Chemical synthesis of a human interferon-alpha 2 gene and its expression in Escherichia coli

A 511-base pair DNA fragment encoding human interferon-alpha 2 has been chemically synthesised and expressed from a lac UV5 and a synthetic trp promoter in Escherichia coli. The synthesis involved preparation of 68 oligodeoxyribonucleotides and their enzymic ligation. The product expressed from the trp promoter system had high antiviral activity and displayed biological effects similar to those of Namalwa interferon on natural killer cell activity and in a Daudi cell growth inhibition assay. E.coli minicells containing plasmid DNA with the synthetic IFN-alpha 2 gene under trp promoter control produce a protein with the same electrophoretic mobility as a sample of authentic IFN-alpha 2. The protein from E.coli cross-reacts with the monoclonal antibody NK-2 and was readily purified, close to homogeneity, by immunoadsorption chromatography on NK-2 sepharose.     

Synchronous growth and genome replication in the blue-green alga Anacystis nidulans

Summary Following the induction of synchronous growth of Anacystis nidulans by light and CO₂ deprivation, cell mass and RNA and DNA content during two cell cycles were measured. Both RNA and DNA synthesis were discontinuous and marked variation in survival to ultraviolet light was related to the state of replication. A model is presented which accounts for the proportion of cells (66%) induced into synchronous genome replication which is also related to the state of replication at the onset of pre-synchrony treatment.