Adenosine Triphosphatase Activity at the External Surface of Chicken Brain Synaptosomes

An ATP-hydrolysing activity on the external surface of intact synaptosomes from chicken forebrain has been investigated. The observed ATPase activity was not due to leakage of the intracellular ATPase activities, of artefacts resulting from breakage of the nerve endings during the incubation and isolation periods, or to possible contamination by other subcellular particles. Disruption of the synaptosomes resulted in an approximately 2.5-fold increase of the basal, Mg2+-dependent ATPase activity, suggesting that the plasma membrane was acting as permeability barrier to the substrate. ATP hydrolysis was maximal (0.8 mumol Pi/min/mg protein) at pH 8.2 in a medium containing either Mg2+ or Ca2+ ions. Ouabain (0.2 mM) and oligomycin (2 micrograms/mg protein) had no appreciable effect on this ATPase activity. Kinetic studies of the enzyme revealed an apparent Km value of ATP of approximately 4 x 10(-5) M. These data are consistent with the view that the observed ATP hydrolysis was being catalysed by an ectoenzyme, i.e., an enzyme in the plasma membrane of the nerve endings with its active site facing the external medium. The rapid hydrolysis of the released ATP is a suspected function for this ecto-ATPase.     

Light-limited growth of two strains of the marine diatom Phaeodactylum tricornutum Bohlin: Chemical composition,carbon partitioning and the diel periodicity of physiological processes

Two isolates of the marine pennate diatom Phaeodactylum tricornutum Bohlin were grown in semi-continuous, nutrient-sufficient culture at varying irradiances on a 12-h light, 12-h dark illumination cycle. The reponse of the isolates to varying degrees of light limitation differed with respect to all of the compositional parameters measured, including growth rates, elemental composition, chlorophyll content, and the partitioning of cellular carbon into four biochemical classes: proteins, lipids, polysaccharides, and low-molecular weight intermediates. The isolates also differed with respect to the relative contributions of light-period and dark-period uptake to the total uptake of ammonium and phosphate ions, although in all cases uptake took place at a reduced rate in the dark. They did not differ with respect to the diel periodicity of cell division, chlorophyll synthesis, and biochemical synthesis. Slightly more cell division took place during the dark period than during the light period. The specific rate of chlorophyll synthesis in the light period, when expressed as a function of irradiance, saturated rapidly; the rate was nearly constant for all irradiances > 100 βE · m^?2 · s^?1. Chlorophyll synthesis in the dark was positively correlated with irradiance over the entire range of irradiances, except where photoinhibition was involved. Protein was synthesized in both the light and dark periods, but at a reduced rate in the dark. Polysaccharides were synthesized during the light period and consumed during the dark period. Lipids and low molecular weight intermediates were synthesized during the light period, but showed little net change during the dark period.     

Photoinactivation of Ammonia Oxidation in Nitrosomonas

Photoinactivation of ammonia oxidation in cells of Nitrosomonas was shown to follow first-order kinetics with a rate constant proportional to incident light intensity. The action spectrum for photoinactivation consisted of a broad peak in the ultraviolet range, where both hydroxylamine and ammonia oxidation were affected, and a shoulder at approximately 410 nm where only ammonia oxidation was affected. In photoinactivated cells, hydroxylamine but not ammonia was oxidized to nitrite and hydroxylamine but not ammonia caused reduction of cytochromes in vivo. The amount per cell of the following constituents was not measurably altered by photoinactivation: cytochromes b, c, a, and P460; ubiquinone; phospholipid; free amino acids; hydroxylamine-dependent nitrite synthetase; nitrite reductase; p-phenylenediamine oxidase; and cytochrome c oxidase. Malonaldehyde or lipid peroxides were not detected in photoinactivated cells. Photoinactivation was prevented (i) under anaerobic conditions, (ii) in the presence of methanol, allylthiourea, thiosemicarbazide, hydroxylamine, ethylxanthate, or CO at concentrations wich caused 100% inhibition of ammonia oxidation, and (iii) at concentrations of ammonia or hydroxylamine which gave a rapid rate of nitrite production. Recovery of ammonia oxidation activity in 90% inactivated cells took place in 6 h, required an energy and/or nitrogen source, and was inhibited by 400 mug of chloramphenicol per ml.     

Inhibition by local anaesthetics of adenine nucleotide translocation in rat liver mitochondria

1. The mechanism of adenine nucleotide translocation in mitochondria isolated from rat liver was further examined by using the local anaesthetics procaine, butacaine, nupercaine and tetracaine as perturbators of lipid-protein interactions. Each of these compounds inhibited translocation of ADP and of ATP; butacaine was the most effective with 50% inhibition occurring at 30mum for 200mum-ATP and at 10mum for 200mum-ADP. The degree of inhibition by butacaine of both adenine nucleotides was dependent on the concentration of adenine nucleotide present; with low concentrations of adenine nucleotide, low concentrations of butacaine-stimulated translocation, but at high concentrations (greater than 50mum) low concentrations of butacaine inhibited translocation. Butacaine increased the affinity of the translocase for ATP to a value which approached that of ADP. 2. Higher concentrations of nupercaine and of tetracaine were required to inhibit translocation of both nucleotides; 50% inhibition of ATP translocation occurred at concentrations of 0.5mm and 0.8mm of these compounds respectively. The pattern of inhibition of ADP translocation by nupercaine and tetracaine was more complex than that of ATP; at very low concentrations (less than 250mum) inhibition ensued, followed by a return to almost original rates at 1mm. At higher concentrations inhibition of ADP translocation resulted. 3. That portion of ATP translocation stimulated by Ca(2+) was preferentially inhibited by each of the local anaesthetics tested. In contrast, inhibition by the anaesthetics of ADP translocation was prevented by low concentrations of Ca(2+). 4. The data provide further support for our hypothesis that lipid-protein interactions are important determinants in the activity of the adenine nucleotide translocase in mitochondria.     

Effects of negative air ions,noise, sex and age on maze learning in rats

An equal number of male and female rats of the King-Holtzman hybrid breed, divided into two age groups (group A₁ : 21–30 days old and group A₂ : 90–100 days old), were exposed to two levels of noise: N₀ = 30 db and N₁ = 90 db, (Ref. 0.0002μ bar), and three levels of negative air ions: I_O = no measurable ion concentration, I₁ = 7 × 10⁶ ions/cm³ and I₂ = 7 × 10⁷ ions/cm³. Time and error scores of 240 rats running a modified Lashley left-right maze with an escape-from-water motive served as criteria. A randomized complete blocks design with replications (2×3×2×2×10) was selected for treatment by analysis of variance. The results indicate that (a) the males show significantly lower error score in negatively ionized air; and (b) the females swim significantly faster than males under all investigated conditions with no apparent effect of noise or ions on their performance.

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Zusammenfassung Eine gleiche Anzahl m?nnlicher und weiblicher Ratten (n = 120) des King-Holtzman Stammes, unterteilt in 2 Altersgruppen (21–30 und 90–100 Tage) wurden zwei Ger?uschintensit?ten (30 db und 90 db) und 3 Konzentrationen negativer Luftionen I₀: nicht messbar niedrig, I₁:7×10⁶, I₂:7×10⁷ Ionen/cm³ ausgesetzt. Die Wirkung wurde geprüft an der Schwimmzeit und der Anzahl Fehler der Tiere im Lasley-Links-Rechts-Labyrinth. Das Motiv zum Schwimmen war, aus dem Wasser zu entkommen. Die Auswertung durch Varianzanalyse erfolgte unter Nachbildung randomisierter kompletter Blockentwürfe. Die Ergebnisse zeigen, dass (a) die M?nnchen bei negativ ionisierter Luft signifikant weniger Fehler machten und (b) die Weibchen unter allen Bedingungen schneller schwammen als die M?nnchen, ohne dass das L?rmniveau oder die Luftionisation darauf einen Einfluss hatten.

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Resume Un nombre égal de rats males et femelles (120 de chaque) de la race King-Holtzman futdivisé en deux groupes d'age (21 à 30 jours et 90 à 100 jours). Ils furent exposés à 2 intensités auditives (30 et 90 db) et à 3 concentrations d'ions négatifs: I₀ non mesurables (faible), I₁ : 7 × 10⁶ et I₂: 7 × 10⁷ ions/cm³. Les effets d'un tel traitement furent examinés au moyen de deux critères: le temps pris pour sortir de l'eau et le nombre de fautes commises dans un labyrinthe droitegauche de Laslay. Pour l'analyse des variances, on a adopté une répartition due au hasard avec répétitions (2×3×2×2×10). On peut en conclure que: (a) les males font moins d'erreurs dans l'air ionisé négativement et cela de fa?on significative et (b) les femelles ont nagé plus vite que les males dans toutes les conditions de l'essai. Ni le bruit ni le degré d'ionisation ne semblent avoir d'influence sur leur comportement.

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This study was supported in part by a grant from the Faculty Research Fund of the University of Oklahoma.     

Effects of direct electric fields,noise, sex and age on maze learning in rats

Equal numbers of male and female rats of the King-Holtzman hybrid breed, divided into two age groups (group A₁: 21–30 days old and group A₂: 90–100 days old), were exposed to two levels of noise: N₀ = 30 db and N₁ = 90 db, (ref. 0.002µbar), and three levels of direct electric field intensities: E₀ = electric field strength of zero; E₁ = 1,600 v/m; and E₂ = 16,000 v/m. Time and error scores of 240 rats running a modified Lashley left-right maze with an escape-from-water motive served as criteria. A randomized complete blocks design with replications (2 × 3 × 2 × 2 × 10) was selected for treatment by analysis of variance. The results indicate that (a) adult rats pre-exposed to electric fields showed significantly lower error scores than young rats, regardless of sex or the noise condition; (b) after exposure to electric fields all rats swam faster than their controls; (c) after exposure to noise females showed significantly higher time scores than males under the same conditions, regardless of age; and (d) when subjected to the combined effect of electric fields and noise males showed significantly lower time scores than females under the same conditions, regardless of age.

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Zusammenfassung Zwei Altersgruppen (21–30 und 90–100 Tage) männlicher and weiblicher Ratten des King-Holtzman-Stammes wurden 2 Lärmintensitäten (30 db und 90 db) und 3 elektrischen Feldstärken (0, 1.600 v/m und 16.000 v/m) ausgesetzt und die Lernfähigkeit beim Schwimmen im modifizierten Lashley linksrechts Maze geprüft. Das Kriterium waren die Zeit und die Anzahl Fehler bei dem Versuch aus dem Wasser zu entkommen. Auswertung der Messungen durch Varianzanalyse unt er Nachbildung kompletter Blockentwürfe. Die Ergebnisse zeigen: (a) Erwachsene Ratten, die vorher elektrischen feldern ausgesetzt wurden, wiesen signifikant weniger fehler auf als junge Ratten, unabhängig vom Geschlecht und Lärmbedingungen; (b) nach der Exponierung in elektrischen Feldern schwammen alle Ratten schneller als die unbehandelten Kontrollen; (c) nach Lärmeexponierung zeigten weibliche Ratten signifikant höhere Trefferzeiten als Männchen unter den gleichen Bedingungen ohne Beziehung zum Alter und (d) bei gleichzeitiger Anwendung von elektrischen Feldern und Lärm wiesen die Männchen niedrigere Trefferzeiten auf als die Weibchen. Alter spielte dabei keine Rolle.

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Resume Des rats de la race King-Holzman, mâles et femelles, de deux groupes d'âge (21 à 30 et 90 à 100 jours) ont été exposes à deux intensités accoustiques (30 et 90 db) et à 3 champs électriques (0, 1.600 et 16. 000 v/m). On a alors examiné leur capacité d'apprendre à nager dans un labyrinthe droite-gauche de Lashley modifié. Les critères d'examen furent le temps et le nombre de fautes commises pour sortir de l'eau. Les observations furent ensuite traitées par l'analyse de variances en tenant compte de répétitions (2 × 3 × 2 × 2 × 10). Les résultats montrent: (a) Les rats adultes préalablement exposés à des champs électriques on fait moins fautes que de jeunes individus et cela de façon significative quels que soient le sexe ou les conditions de bruit; (b) après avoir été exposés à des champs électriques, tous les rats ont nagé plus vite que les bêtes de contrôle; (c) après exposition au bruit, les femelles ont présenté des temps de sortie significativement plus élevés que les mâles pris dans des conditions identiques et cela quel qu'en soit l'âge et (d) après exposition simultanée au bruit et aux champs électriques, les mâles se sont trouvés moins habiles que les femelles, sans incidence de l'âge.

     

Methacarn (methanol-Carnoy) fixation

Summary According to chemical data, methanol raises the shrinkage temperature of collagen significantly more than ethanol (86° C versus 70° C). Since increase of shrinkage temperature appears desirable in tissues to be embedded in paraffin, methanol was substituted for ethanol in Carnoy's fluid. This methanol-Carnoy mixture is referred to as methacarn solution. The fixation-embedding procedure was similar to that described in the study of Carnoy fixation. Methacarn-fixed sections showed little or no shrinkage and compared well with material fixed in Carnoy's or Zenker's fluid. Myofibrils, especially in endothelial and epithelial cells, were more prominent in methacarn- than in Carnoy-fixed tissues.A review of the chemical literature showed that methanol, ethanol and chloroform stabilize or even enhance helical conformations of proteins, presumably by strengthening of hydrogen bonds. Interference with hydrophobic bonds causes unfolding and/or structural rearrangements in globular proteins. The twin-helical structure of DNA collapses in alcoholic solutions. Hence, methacarn fixation can be expected to preserve the helical proteins in myofibrils and collagen, but the conformations of globular proteins and DNA will be significantly altered. Literature on conformational effects produced by fixatives used in electron microscopy was also reviewed. Glutaraldehyde and OsO₄ cause considerable loss of helix (22–29% and 39–66% respectively). KMnO₄ and glutaraldehyde followed by OsO₄ produce extensive transitions from helical to random-coil conformations similar to those seen in powerful denaturants such as 8 M urea. Evidently these fixatives are unsuitable for studies of helical proteins. In contrast ethylene glycol preserves helical conformations.     

Effects of a Single Histoplasmin Skin Test on the Serological Diagnosis of Histoplasmosis

Numerous reports have indicated that a single histoplasmin skin test may stimulate humoral antibodies to Histoplasma capsulatum antigens in histoplasmin-hypersensitive individuals. Although these investigations concur that antibody elevations are evoked, they vary in the reported degree of incidence and response induced, and they cast doubt on the interpretation of serological tests in the diagnosis of histoplasmosis. Histoplasmin-hypersensitive subjects (114) were bled prior to administration of the skin test, 2 days later, at the time this test was read, and 15 and 30 days after testing. No significant antibody titers were observed at 2 days. At 15- and 30-day intervals, only 17 (15%) of the subjects demonstrated circulating antibodies. All 17 showed agar gel bands; 5 demonstrated no complement-fixation (CF) titers, 10 produced CF antibodies ranging from 1:8 to 1:16, and 2 demonstrated titers of 1:32. The data suggest that skin testing does not interfere significantly with antibody levels in sera drawn approximately 2 days after administration of antigen. However, since titers as high as 1:32 were obtained at later intervals, such reactions should be evaluated cautiously and only after consideration of clinical findings.     

Amino acid compostion of walls from single and filamentous cells of Clostridium acidiurici

Gaffar, Abdul (Brigham Young University, Provo, Utah), David R. Terry, and Richard D. Sagers. Amino acid composition of walls from single and filamentous cells of Clostridium acidiurici. J. Bacteriol. 91:1618-1624. 1966.-The walls from single and filamentous cells of Clostridium acidiurici were shown to contain 11 amino acids: aspartic acid, serine, glutamic acid, proline, d-alanine, glycine, valine, methionine, valine, leucine, phenylalanine, and lysine. In the walls from cells grown at 37 C, d-alanine was the amino acid present in largest quantity, but in the walls from cells grown at 44 C there was a 50% reduction in the d-alanine content while the levels of the other amino acids were unchanged. Filamentous cells grown at 44 C, then brought to 37 C and transferred to fresh medium, fragmented into short cells within 30 min. Alanine racemase activity was the same in extracts from cells grown at both 37 and 44 C, suggesting that this enzyme was not the major controlling factor in the low content of d-alanine in filaments grown at 44 C. Spent medium from cultures grown at 44 C contained a significant amount of d-alanine, whereas there was no evidence of this amino acid in the spent medium from cultures grown at 37 C.     

A comparison of sperm morphology and silver nitrate staining characteristics in the domestic ferret and the black-footed ferret

Ejaculated sperm from the domestic ferret (Mustela putorius furo) and the black-footed ferret (Mustela nigripes) were compared for differences in morphological abnormalities and argentophilic protein distribution. Thawed domestic ferret sperm was also compared to fresh sperm to determine whether there were any effects on cell morphology due to cryopreservation. There were statistically significant differences between the two species of ferret in two of the categories scored. The domestic ferret had a higher frequency of cells that were bent in the midpiece and in the principal piece, and a higher frequency of headless and tailless cells when compared to the black-footed ferret. There were no statistically significant differences in cell morphology between the fresh and cryopreserved ejaculates of the domestic ferret employing a standard egg yolk cryoextender. Silver nitrate staining distribution was different between the two species in both the head and tail region.