Rapid determination of DNA synthesis in adherent cells grown in microtiter plates

A specific, rapid, and economical method for measuring the extent of DNA synthesis in adherent rat hepatoma H4-II-E cells grown in 96-well microtiter plates is described. The adherent cells were pulsed for 1 h with [methyl-3H]thymidine, released from the substratum by trypsinization, and collected on fiberglass filters with a MASH II cell harvester. The amount of radioactivity incorporated was directly proportional to the number of cells per well. Growth curves generated by measuring [methyl-3H]thymidine incorporation and counting the number of cells per well were identical. Experiments with inhibitors of DNA, protein, and RNA synthesis demonstrated that this method selectively measured DNA synthesis. In addition, [3H]thymidine uptake showed excellent correlation with autoradiographic assessment of DNA synthesis. This specific and sensitive method for determining DNA synthesis in microtiter cultures should facilitate studies of effects of various growth-controlling agents on epithelial, fibroblastic, and other cells which grow as adherent cells in culture.     

Solubilization of an Adenosine Uptake Site in Brain

Procedures are described for the solubilization of adenosine uptake sites in guinea pig and rat brain tissue. Using [3H]nitrobenzylthioinosine [( 3H]NBI) the solubilized site is characterized both kinetically and pharmacologically. The binding is dependent on protein concentration and is saturable, reversible, specific, and high affinity in nature. The KD and Bmax of guinea pig extracts are 0.13 +/- 0.02 nM and 133 +/- 18 fmol/mg protein, respectively, with linear Scatchard plots obtained routinely. Similar kinetic parameters are observed in rat brain. Adenosine uptake inhibitors are the most potent inhibitors of [3H]NBI binding with the following order of potency, dilazep greater than hexobendine greater than dipyridamole. Adenosine receptor ligands are much less potent inhibitors of binding, and caffeine is without effect. The solubilized adenosine uptake site is, therefore, shown to have virtually identical properties to the native membrane site. The binding of the adenosine A1 receptor agonist [3H]cyclohexyladenosine [( 3H]CHA) to the solubilized brain extract was also studied and compared with that of [3H]NBI. In contrast to the [3H]NBI binding site [3H]CHA binds to two apparent populations of adenosine receptor, a high-affinity site with a KD of 0.32 +/- 0.06 nM and a Bmax of 105 +/- 30 fmol/mg protein and a lower-affinity site with a KD of 5.50 +/- 0.52 nM and Bmax of 300 +/- 55 fmol/mg protein. The pharmacology of the [3H]CHA binding site is consistent with that of the adenosine receptor and quite distinct from that of the uptake [( 3H]NBI binding) site. Therefore, we show that the adenosine uptake site can be solubilized and that it retains both its binding and pharmacologic properties in the solubilized state.     

Chemically induced vasospasm: the effect of ischemia, vessel occlusion, and adrenergic blockade

Vasospasm in revascularized tissue can compromise tissue perfusion even though the microsurgical anastomoses remain patent. Circulating catecholamine stimulates peripheral vasoconstriction. Chemical vasospasm was induced by the intraarterial administration of norepinephrine to denervated rat hind limbs. Heel pad blood flow was assessed by laser-Doppler velocimetry. Mean blood flow was 463 +/- 106 in the denervated leg and 337 +/- 50 in the opposite (intact) leg (p less than 0.01). Flow in the denervated leg decreased 78 percent (463 +/- 106 to 100 +/- 23) within 5 minutes of norepinephrine administration and did not return to normal for 30 minutes. Norepinephrine administration in the presence of 1 and 3 hours of ischemia decreased flow at 5 minutes to 6.6 and 6.0 percent of normal, respectively (31 +/- 14, 28 +/- 14, control 100 +/- 23; p less than 0.001). Administration of intraarterial norepinephrine distal to a femoral artery occluded for 1 and 5 minutes reduced flow following clamp release to 11.2 and 7.1 percent of normal 5 minutes after clamp release (52 +/- 9, 33 +/- 7, control 100 +/- 23; p less than 0.001). Comparison of the 1-minute and 5-minute groups to each other showed a significant flow decrease in the 5-minute group (p less than 0.007). This indicates that the observed decrease in flow was related both to the presence of a vessel occlusion and to the length of the occlusion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biology and defensive adaptations in Rodatus major (Coleoptera: Coccinellidae) and its prey, Monophlebulus pilosior (Hemiptera: Margarodidae)

The biology of the Australian coccinellid Rodatus major (Blackburn) and its prey, the hemipteran margarodid Monophlebulus pilosior (Maskell), are described. New predators and a new parasite of M. pilosior are recorded. Rodatus major makes use of elaborate and distinctive defensive adaptations and behaviour to protect itself from predators. They include cryptic coloration, aposematic behaviour, production of wax by larvae, production of a large, thick protective covering concealing the pupa and resembling an M. pilosior ovisac, and reflex bleeding. It is the only known coccinellid species in which both prepupa and pupa are hidden by a protective shroud of wax threads. Rodatus major has a specialized feeding behaviour. Its potential as a biological control agent is assessed. It is only the second Australian margarodid-feeding coccinellid to be studied, Rodolia cardinalis (Mulsant) being the first.     

Increase of translational fidelity blocks sporulation in the fungus Podospora anserina

Summary AS7-1 and AS7-2 are antisuppressor mutations reducing the miscoding capacity of ribosomes. Strains carrying and AS7 mutation do not sporulate. We have investigated whether the sporulation deficiency is due to the decrease of translational ambiguity. Two major findings argue in favour of this assumption. First, a significant sporulation level is restored in the presence of paromomycin. Second, three mutations which restore the sporulation of AS7-2 increase the ribosomal misreading in vitro. They define two new loci for ribosomal suppressors, su11 and su12. The two ribosomal proteins altered by su11-1 and su12-1 have been identified by electrophoresis. The results are discussed in the context of a more general hypothesis proposed by Picard-Bennoun (1982).     

Response of end bud cells from immature rat mammary gland to hormones when cultured in collagen gel

End buds from 4- to 5-week-old rat mammary glands were isolated and cultured within a rat tail tendon collagen gel matrix. Media containing equine serum or porcine serum and cholera toxin promoted growth, but not the production of casein or thioesterase II, nor did they induce a state of differentiation as assessed by cell ultrastructure. Medium supplemented with only 5% porcine serum, insulin and cholera toxin did not support growth or differentiation. However, when prolactin, estradiol, progesterone and hydrocortisone were added to this medium, growth was stimulated greatly and a differentiated state was induced as assessed by the production of casein and thioesterase and by the appearance of a highly secretory ultrastructure.