Dissociation by Chelating Agents and Substructure of the Thermophilic Bacteriophage TP84

The thermophilic bacteriophage TP84 is dissociated into its head, tail, and released deoxyribonucleic acid (DNA) by chelating agents such as ethylenediaminetetraacetic acid (EDTA) and phosphate. The phage is more sensitive to EDTA than to phosphate, and dialysis against either agent causes more effective dissociation than standing in their presence. The tail possesses a knobbed structure which is inserted into the head of the intact phage and to which the DNA appears to be attached. The method of dissociating TP84 described in this paper provides a source of undamaged structural components and intact strands of DNA for subsequent investigations. A possible mechanism of chelate inactivation is discussed.     

Enzyme-Substrate Reactions in Very High Magnetic Fields. II

We have examined the effect of conditioning the enzyme trypsin in solution at pH 8.2 in a large magnetic field before determining its reactivity towards a synthetic substrate N-benzoyl DL-arginine p-nitroanilide (BAPA). This "pretreatment" was allowed to proceed for as long as 3(2/3) hr in a magnetic field of 208 kgauss at temperatures 26 and 36 degrees C. No effect on reactivity was observed when such pretreated enzyme solutions were compared with identical but untreated enzyme solutions. A single such reaction, allowed to proceed directly in a magnetic field of 220 kgauss for 9 min, similarly showed no difference in rate from its control.     

CYTOPLASMIC DNA SYNTHESIS IN AMOEBA PROTEUS : I. On the Particulate Nature of the DNA-Containing Elements

The incorporation of tritiated thymidine in Amoeba proteus was reinvestigated in order to see if it could be associated with microscopically detectable structures. Staining experiments with basic dyes, including the fluorochrome acridine orange, revealed the presence of large numbers of 0.3 to 0.5 µ particles in the cytoplasm of all cells studied. The effect of nuclease digestion on the dye affinity of the particles suggests that they contain DNA as well as RNA. Centrifugation of living cells at 10,000 g leads to the sedimentation of the particles in the centrifugal third of the ameba near the nucleus. Analysis of centrifuged cells which had been incubated with H₃-thymidine showed a very high degree of correlation between the location of the nucleic acid-containing granules and that of acid-insoluble, deoxyribonuclease-sensitive labeled molecules and leads to the conclusion that cytoplasmic DNA synthesis in Amoeba proteus occurs in association with these particles.     

Abnormal Intranuclear and Cytoplasmic Formations Associated with a Chemically Induced, Transplantable Chicken Sarcoma

Thirty GRCH/15 tumors (a 1, 2, 5, 6-dibenzanthracene-induced chicken sarcoma) were examined in the light and the electron microscope. Associated with the sarcoma were two types of abnormal intranuclear lesions, one in the form of a vacuole, the other as an aggregate containing glycogen. In the electron microscope, one type of lesion observed showed an organized microfibrillar structure. Abnormal cytoplasmic formations occurred as massed clusters of thread-like or tubular material, which gave rise to small bodies with concentric shell structure; similar bodies were found associated with vacuoles.     

Free Radical Mediated Cytotoxicity of Desferrioxamine

Toxic effects of desferrioxamine (DFO) upon cell growth were assayed with continuous bromodeoxyur-idine (BrdU) labeling and bivariate ethidium bromide/Hoechst 33258 Row cytometry. At 5% oxygen DFO caused a dose-dependent inhibition of cell growth. which was potentiated at 20% oxygen. and by cumene hydroperoxide but not by paraquat. An irreversible arrest in the GZ phase of the cell cycle was the cell-kinetic mechanism underlying this growth inhibition. The G2 arrest was not dependent upon the BrdU concentration in the medium, thus ruling out a direct attack of a free radical on thymidine residues. The observed cytotoxicity of DFO cautions against its use in the treatment of conditions of elevated oxidative stress.     

Probenicid inhibition of fluorescence extrusion after MCB-staining of rat-1 fibroblasts

The intracellular fluorescence level of cells stained continuously with monochlorobimane was monitored by flow cytometry in order to assess the initial rate of glutatione to monochlorobimane conjugation as a measure of glutathione S-transferase activity. In addition to a rapid initial increase and a plateau level, a decline in fluorescence intensity was found upon prolonged flow cytometric monitoring. Exposure to probenicid, an inhibitor of an ATP-dependent organic anion pump, prevented this decrease. Incubation with vanadate and verapamil was without effect. Thus, extrusion of fluorescentglutathione-conjugate perturbs the proportionality between initial glutathione level and monochlorobimane-dependent fluorescence intensity. Monitoring by flow cytometry the decrease in monochlorobimane-dependent fluorescence may be useful to detect multidrug resistant cells.     

Autoradiographic comparison of [I]LSD-labeled 5-HT2A receptor distribution in rat and guinea pig brain

Although the density and distribution of 5-HT_2A(5-hydroxytryptamine-2A) receptors is well established for rat brain, the 5-HT_2A receptor distribution and density in guinea pig brain has not been extensively studied. In the present in vitro study, we have utilized ¹²⁵I-lysergic acid diethylamide ([¹²⁵I]LSD) to quantify and compare 5-HT_2A receptor density in coronal sections of rat and guinea pig brain. Spiperone (1 μM) and sulpiride (1 μM) were used to displace [¹²⁵I]LSD binding from 5-HT_2A and D₂ binding sites, respectively. Ligand binding was quantified by computer-aided image analysis densitometry (MCID). Similar to the rat, areas of highest specific 5-HT_2A receptor binding (fmol/mg protein) in guinea pig brain included the claustrum and Layer 4 of the cerebral cortex. Significant binding was also found in remaining neocortical layers, islands of Calleja, caudate putamen, olfactory bulb, nucleus accumbens, and choroid plexus. While the rat brain exhibited a high level of specific binding in the tenia tecta and mammillary nuclei, little binding was observed in these regions in the guinea pig. In both rat and guinea pig, low specific binding was found in amygdaloid, thalamic, or cerebellar areas. These studies indicate a general similarity between 5-HT_2A binding site distribution and relative density in guinea pig and rat brain but point to a few brain regions where significant differences exist.     

Associations of esterase 6 allozyme and activity variation with reproductive fitness inDrosophila melanogaster

Previous studies have shown that the esterase 6 (EST6) enzyme ofD. melanogaster is mainly produced in the sperm ejaculatory duct of the adult male and comparisons of wild-type males with laboratory null mutants have suggested that the enzyme plays a role in reproductive fitness. In this study we have compared 18 field-derived lines each isoallelic forEst6 for differences in five components of male reproductive fitness. No consistent fitness differences were found among lines differing in respect of the two major allozyme classes EST6-F and EST6-S, despite other evidence that these two classes are not selectively equivalent in the field. However, differences in reproductive fitness were found among lines differing in the minor mobility variants that segregate within EST6-F and EST6-S. A failure to distinguish among these minor forms may explain the discrepancies in previous studies on the effects of the major EST6 allozymes on reproductive fitness. The most significant associations we have found between EST6 and reproductive fitness were due to variation in EST6 activity levels. Male EST6 activity levels were found to be positively correlated with their time to first mating, negatively correlated with the numbers of eggs laid and progeny produced by their mates, and negatively correlated with the frequency with which their mates remate. We conclude that some EST6 variants differ in components of male reproductive fitness operative in laboratory cultures. However, the evidence for fitness differences is stronger for variants affecting the amount, rather than the structure of the enzyme, and the direction of the differences varies between some of the fitness components tested.     

Disruption of Potential α-Helix in the G Loop of the Guinea: Pig 5-Hydroxytryptamine2 Receptor Does Not Prevent Receptor Coupling to Phosphoinositide Hydrolysis

Abstract: Heterogeneity of the 5-hydroxytryptamine₂ (5-HT₂) receptor across species has been implicated in several pharmacological and physiological studies. Although 5-HT₂ receptors in the rat have been linked to increases in Phosphoinositide (PI) hydrolysis, little evidence exists to support the association of guinea pig 5-HT₂ receptors with Pl hydrolysis, the second messenger generally linked with 5-HT₂receptors. In the present study, we have taken a molecular and biochemical approach to determining whether species differences in brain 5-HT₂ receptors exist between rat and guinea pig. First, we isolated partial cortical 5-HT_a receptor cDNA clones that encompassed the third intracellular loop, a receptor area putatively important in receptor-effector coupling. The amino acid sequences deduced from the cDNA clones for rat and guinea pig brain 5-HT₂ receptor were 97% homologous. However, the guinea pig 5-HT₂ receptor had two tandem substitutions that disrupted a potential alpha helix in the region of the third cytoplasmic loop, which theoretically could alter the intracellular coupling of the guinea pig cortical 5-HT₂ receptor. Because of these molecular differences, we examined further the pharmacological activation of the brain 5-HT₂ receptor from guinea pig. 5-HT and the 5-HT₂ receptor agonist α-methyl-5-HT increased PI hydrolysis in guinea pig cortical slices whereas the 5-HT_1c receptor agonist 5-methyltryptamine was significantly less potent. In addition, the 5-HT₂ receptor antagonists LY53857, ketanserin, and spiperone blocked 5-HT-stimulated Pl hydrolysis. These pharmacological data suggested that activation of the 5-HT₂ receptor in guinea pig cortical slices was associated with PI hydrolysis. Thus, although areas of the guinea pig brain 5-HT₂ receptor that influence receptor-effector coupling were different from the rat, such differences were not critical to receptor-effector coupling because, as in the rat, guinea pig brain 5-HT₂ receptors were also coupled to PI hydrolysis.     

The direction of mothers' and daughters' preferences and the heritability of male ornaments in red jungle fowl (Gallus gallus)

We used a breeding design involving 18 sires and 108 dams tostudy the heritabilities of male ornaments in red jungle fowl(Gallus gallus). Ornaments used by females to choose mates showedlow heritabilities, with the exception of comb and wattle measures.The general absence of heritability suggests that a geneticcovariance did not exist at the time of this study between mostmale ornaments and female preferences for those ornaments. Thisresult is contrary to a key prediction of the arbitrary or Fisherianhypothesis of sexual selection. Comb size and color are condition-dependenttraits that reflect short-term changes in health, and comb sizeof males was positively correlated with offspring weight. Ourresults are consistent with the expectation of good-genes hypothesesthat male ornaments reflect the ability of males to withstandenvironmental stresses.