On the mechanism of photosynthetic electron transfer in Rhodopseudomonas capsulata and Rhodopseudomonas sphaeroides

(1) Current models for the mechanism of cyclic electron transport in Rhodopseudomonas sphaeroides and Rhodopseudomonas capsulata have been investigated by observing the kinetics of electron transport in the presence of inhibitors, or in photosynthetically incompetent mutant strains. (2) In addition to its well-characterized effect on the Rieske-type iron sulfur center, 5-(n-undecyl)-6-hydroxy-4,7-dioxobenzothiazole (UHDBT) inhibits both cytochrome b₅₀ and cytochrome b_?90 reduction induced by flash excitation in Rps. sphaeroides and Rps. capsulata. The concentration dependency of the inhibition in the presence of antimycin (approx. 2.7 mol UHDBT/mol reaction center for 50% inhibition of extent) is very similar to that of its inhibition of the antimycin-insensitive phase of ferricytochrome c re-reduction. UHDBT did not inhibit electron transfer between the reduced primary acceptor ubiquinone (Q^?_I) and the secondary acceptor ubiquinone (Q_II) of the reaction center acceptor complex. A mutant of Rps. capsulata, strain R126, lacked both the UHDBT and antimycin-sensitive phases of cytochrome c re-reduction, and ferricytochrome b₅₀ reduction on flash excitation. (3) In the presence of antimycin, the initial rate of cytochrome b₅₀ reduction increased about 10-fold as the E_h(7.0) was lowered below 180 mV. A plot of the rate at the fastest point in each trace against redox potential resembles the Nernst plot for a two-electron carrier with E_m(7.0) ≈ 125 ± 15 mV. Following flash excitation there was a lag of 100–500 μs before cytochrome b₅₀ reduction began. However, there was a considerably longer lag before significant reduction of cytochrome c by the antimycin-sensitive pathway occurred. (4) The herbicide ametryne inhibited electron transfer between Q^?_I and Q_II. It was an effective inhibitor of cytochrome b₅₀ photoreduction at E_h(7.0) 390 mV, but not at E_h(7.0) 100 mV. At the latter E_h, low concentrations of ametryne inhibited turnover after one flash in only half of the photochemical reaction centers. By analogy with the response to o-phenanthroline, it is suggested that ametryne is ineffective at inhibiting electron transfer from Q^?_I to the secondary acceptor ubiquinone when the latter is reduced to the semiquinone form before excitation. (5) At E_h(7.0) > 200 mV, antimycin had a marked effect on the cytochrome b₅₀ reduction-oxidation kinetics but not on the cytochrome c and reaction center changes or the slow phase III of the electrochromic carotenoid change on a 10-ms time scale. This observation appears to rule out a mechanism in which cytochrome b₅₀ oxidation is obligatorily and kinetically linked to the antimycin-sensitive phase of cytochrome c reduction in a reaction involving transmembrane charge transfer at high E_h values. However, at lower redox potentials, cytochrome b₅₀ oxidation is more rapid, and may be linked to the antimycin-sensitive reduction of cytochrome c. (6) It is concluded that neither a simple linear scheme nor a simple Q-cycle model can account adequately for all the observations. Future models will have to take account of a possible heterogeneity of redox chains resulting from the two-electron gate at the level of the secondary quinone, and of the involvement of cytochrome b_?90 in the rapid reactions of the cyclic electron transfer chain     

Toxoplasma gondii: lymphocyte function during acute infection in mice

T-cell function during acute Toxoplasma gondii infection was evaluated in murine models. Blastogenic response to the T-cell mitogen concanavalin A (Con A) was not depressed during infection with either the C37 or the C56 strain of T. gondii in either $\text{BALB}\text{c}$ or $\text{C}\text{57}\text{BL}\text{6}\text{J}$ mice that were inoculated either intravenously or intraperitoneally with varying doses of tachyzoites 7, 14, or 30 days earlier. In evaluation of lymphocytes from individual mice, utilization of a range of concentrations of Con A was found to be important for correct interpretation of results. There was variability in the magnitude of response of individual mice and in the concentration of mitogen that produced an optimal response among the inbred mice. The T-cell-dependent, primary antibody response to sheep red blood cells (SRBC) was not depressed in $\text{BALB}\text{c}$ mice infected with the C37 strain of Toxoplasma 1 and 8 days prior to inoculation with SRBC. A lower blastogenic reponse to Con A of lymphocytes from $\text{C}\text{57}\text{BL}\text{6}\text{J}$ mice compared with that of $\text{BALB}\text{c}$ mice appeared to correlate with increased susceptibility of $\text{C}\text{57}\text{BL}\text{6}\text{J}$ mice to low-challenge inocula of T. gondii.     

Primary cultures and the levels of cytochromeP450 in hepatocytes from mouse,rat, hamster,and rabbit liver

Summary Hepatocyte primary cultures (HPC) derived from rat, mouse, hamster, and rabbit liver were characterized for a variety of parameters. The conditions that maximized recovery, attachment, and survival varied between species. Hepatocytes from all four species were capable of attaching in serum-free Williams’ medium E (WME), but optimal attachment as monolayer cultures was achieved for mouse and hamster HPC in medium receiving 1% calf serum supplementation. Hamster hepatocytes required additional cations, whereas rabbit and rat hepatocytes displayed maximal attachment in medium supplemented with 10% calf serum. Survival of mouse and rabbit hepatocytes after 24 h in serum supplemented media was in the order of 90%. Rat and hamster hepatocyte 24 h survival was approximately 70 and 60%, respectively, and was not significantly affected by serum supplementation. Hepatocytes from each species varied in their content of cytochromeP450 at the time of isolation and in the rate of reduction during culture. Mouse and rat hepatocytes demonstrated the most rapid decline in content during the initial 24 h in culture, whereas concentrations in rabbit hepatocytes were virtually unchanged. The rate of decline inP450 concentrations in hamster hepatocytes was intermediate between those displayed by rat and rabbit hepatocytes. These studies have delineated conditions useful for the culture of hepatocytes from four species and have documented the status of an important parameter of their functional capability. This study was supported by EPA contract 68-01-6179. C. J. Maslansky was a recipient of a Monsanto Fund Fellowship in Toxicology.     

The effect of re-release of Oryctes rhinoceros baculovirus in the biological control of rhinoceros beetles in Western Samoa

The Rhinoceros Beetle Project in Western Samoa has developed and successfully applied biological methods to control the rhinoceros beetle, a serious pest of coconut palms, by using two specific pathogens, a baculovirus (Family Baculoviridae), and an entomopathogenic fungus, Metarhizium anisopliae. The application of virus particularly has markedly suppressed the beetle population and helped revive the copra industry. The virus disease had established itself in the wild beetle population several years after its introduction at a level between 30 and 50%. At the same time an increase in beetle numbers and damage to palm trees was experienced. Therefore, a continuous release of virus into beetle-infested areas was proposed. It was argued that, considering the relatively high level of “natural” virus incidence, further releases of virus into the population would be futile. In a combined research and control program, virus was again re-released into the wild beetle population which was already virus infected. The results show that through re-release the virus level can be raised and the number of beetles and consequently the damage can be reduced. The techniques of the control methods are described. The virus release is very easy and cheap; it requires no chemicals, no special equipment, and it is particularly recommended in situations where breeding places are inaccessible or other methods such as plantation sanitation are either impossible or economically impractical. Above all, the methods are absolutely safe from the standpoint of environmental protection.     

A Comparison of the Polypeptide Pattern Between a Brain Primary Culture and Fractions of Bulk-Isolated Glial Cells

Abstract: This paper reports on the electrophoretic protein/polypeptide pattern of a rat brain primary culture. For comparison, the polypeptide pattern of neuronal and glial enriched fractions from adult rat brain and cerebral hemispheres from newborn and adult rat have been analysed. Water-soluble and SDS-extractable polypeptide fractions appeared and/or increased in amount in the cultures until confluency. The polypeptide pattern of the cultures most resembled that of the glial cell fractions, showing some of this fraction's specificity. Removal of fetal calf serum and addition of 0.1 mM dibutyryl cyclic adenosine monophosphate (dB-cAMP) produced few changes in the electrophoretic pattern. The study thus provides evidence in favour of the astroglial nature of the brain primary culture. It also shows that the cells undergo some maturation in the culture.     

COMPARISON OF IN SITU PHOTOSYNTHETIC ACTIVITY OF EPIPHYTIC,EPIPELIC AND PLANKTONIC ALGAL COMMUNITIES IN AN OLIGOTROPHIC LAKE,SOUTHERN FINLAND1

The photosynthetic activity of different algal communities at the outer edge of an Equisetum fluviatile L. stand in an oligotrophic lake (Pääjärvi, in southern Finland) was investigated. Production by the algal communities was measured simultaneously using a modified ¹⁴C-method, and the results were related to the volume of algae and the available irradiance. The relative production rate (P/B quotient) of phytoplankton was ca. 3 × that of epiphyton and ca. 20 × that of epipelon. Epiphyton productivity remained almost constant although the algal volume varied greatly, suggesting that the surface layer of the algal community was mainly responsible for the photosynthetic activity. In the littoral area (at 1 m depth) primary production/m² of lake surface by phytoplankton, epiphyton and epipelon was similar but in the littoriprofundal area (2–4 m) phytoplankton production was twice that of epipelon. Primary productivity of epiphyton and epipelon/m² of substratum was about equal to phytoplankton productivity/m³ of water at the same irradiance. This relation provided a means of estimating the relative contributions of the different algal communities to the total algal production in the lake.     

Tissue specific variation of C-glycosylflavone patterns in oat leaves as influenced by the environment

A comparison is made between the flavone patterns accumulating in epidermal tissues and in the mesophyll of oat primary leaves grown in a phytotron and under field conditions. In developing leaves cultivated under standard conditions, varying patterns of two vitexin-derived O-rhamnosides and of one isovitexin O-arabinoside are produced in the basal region as the result of basal meristem activity. These patterns are tissue specific and differ quantitatively in the epidermis and the mesophyll. During the course of subsequent growth and differentiation, this pattern is constant as the compounds are moved upwards due to basipetal leaf growth. In comparison, the flavone patterns generated in the basal section of leaves grown in the field do not vary significantly. There is the additional accumulation of isoorientin O-arabinoside. Again flavone patterns are tissue specific, but in contrast to standard growth they are modified characteristically in those leaf tissues which are already morphologically differentiated. It is possible that the isovitexin moiety of the O-arabinoside is oxidized to the corresponding isoorientin derivative in the mesophyll. Moreover, field-grown leaves show a two-fold increase in flavone content in each leaf epidermis and a six-fold increase in the mesophyll when compared to the corresponding tissues of phytotron-grown leaves.     

Chemically defined serum-free media for the cultivation of primary cells and their susceptibility to viruses

Summary Chemically defined media SFRE-199-1 for the growth and SFRE-199-2 for the maintenance of primary baboon kidney (Bak) cell cultures were formulated by supplementing medium M199 with insulin, sodium pyruvate, zinc sulfate, and increasing arginine-HCl, cysteine, cystine,l-glutamine,l-glutamic acid, glycine, histidine, tyrosine, and glucose to maximally active nontoxic concentrations. For prolonged maintenance of the cells, physiological pH control, and blocking of excessive lactic acid accumulation in the spent medium of the cell cultures, it was necessary to supplement the medium containing Earle's balanced salts withd-(+) galactose. The cells grew and were maintained equally well on glass or polystyrene surfaces. Selenium, when added to growth medium or substituted for insulin and zinc sulfate, did not stimulate cell growth. Electron microscopy showed that numerous dense particles, approximately 250 to 400 ? in diameter, with the appearance of glycogen, were found throughout the cytoplasm in the cells grown in SFRE-199-1 and maintained in SFRE-199-2. Echovirus types 1 to 3, poliovirus types 1 to 3, coxsackievirus types B2, B4, B5,Herpesvirus hominis type 1, simian herpesvirusH. simiae and SA8, and simian adenovirus SV34 when titrated in primary Bak cells and grown and maintained in SFRE-199-1 and 2, respectively, developed titers comparable to those obtained in conventionally grown and maintained cells. This study was supported in part by National Institute of Health Grant RR00361 and World Health Organization Grant V4/181/38. This laboratory serves as the NIH/WHO Collaborating Center for Reference and Research in Simian Viruses.     

Primary culture of mouse mammary tumor epithelial cells embedded in collagen gels

Summary Mammary tumor epithelial cells from BALB/cfC3H mice were dispersely embedded inside the collagen gels in Ham's F-12 medium containing horse serum. A sustained cell growth leading to a 5- to 10-fold increase in cell number over initial level was observed in less than 2 weeks. The extent of this growth was found to be dependent on serum concentration. However, addition of various protein and steroid hormones, both singly and in combination, to low-serum-containing medium failed to achieve a comparable level of growth to that promoted by higher serum concentration. Mammary tumor cells can now be consistently propagated in primary culture. This investigation was supported by Grants CA05388 and CA09041 awarded by the National Cancer Institute, Department of Health, Education and Welfare, and by cancer research funds of the University of California.     

Hepatocytes from newborn and weanling rats in monolayer culture: Isolation by perfusion,fibronectin-mediated adhesion,spreading, and functional activities

Summary The two-step collagenase perfusion method originally developed for the high yield isolation of parenchymal cells from adult rat livers has been adapted to rats of 1 day, 1 week, and 3 weeks of age. The use of this method to isolate hepatocytes from five or six rats of the respective ages demonstrated its reliability in terms of cell yield, percentage of single cells, and cell viability. In all cases, hepatocytes attach with high efficiency to fibronectin precoated dishes using serum-free culture medium. The dynamics of spreading is faster for newborn hepatocytes than adult ones. The functional integrity of these parenchymal liver cells was assessed by their capacity to secrete albumin and α-fetoprotein in serum-free medium and to express lactate dehydrogenase activity over a 24-hr period in primary culture. Part of this work was presented at the 30th Annual Meeting of the Tissue Culture Association, Seattle, June, 1979.