Elimination of the non-specific binding of avidin to tissue sections

Summary A simple procedure is described for eliminating non-specific staining with avidin—peroxidase conjugates. Murine ovaries were embedded in either paraffin wax or epoxy resin and, after blocking endogenous peroxidase activity, were treated with 10 µg/ml biotinylatedPisum sativum agglutinin. Avidin—peroxidase conjugates (5 µg/ml), diluted in standard 0.05m tris-buffered saline, pH 7.6, containing 0.139m NaCl, produced considerable background coloration and intense mast cell staining in controls without the lectin. This background diminished as the ionic strength of the buffer was raised. At 0.125m Tris-buffered saline (containing 0.347m NaCl) the background was completely unstained, with elimination of all binding to mast cells and only minimal loss of specific lectin binding.     

Die Entwicklung der Laut?u?erungen bei der Graugans (Anser anser)

Zusammenfassung Das Lautrepertoire von sechs handaufgezogenen Graugänsen wurde in individuellen Längsschnitten vom Schlüpfen bis zum Alter von 100 Tagen mit sonagraphischer Methode analysiert. Die Einteilung des Repertoires erfolgte nach drei verschiedenen Kriterien: Klang, Sonagramm und Kontext. Im jugendlichen Repertoire ließen sich 4 Rufklassen voneinander trennen. Im Laufe der Jugendentwicklung veränderten sich vor allem die phonetischen Parameter der Rufe, während die syntaktischen weitgehend konstant blieben. Nach einem Anstieg in der ersten Woche fiel die Tonhöhe der Rufe bei allen untersuchten Individuen und Rufklassen gleichmäßig ab. Der Stimmbruch, meßbar an der Zunahme geräuschhafter Komponenten, war ein kontinuierlicher, langfristiger Prozeß. Die Individualität war nicht in einem einzelnen, wohl aber in der Kombination mehrerer Merkmale faßbar. Die Geschlechter waren schon früh an Unterschieden in der Tonhöhe erkennbar. An sonstigen ontogenetischen Veränderungen innerhalb des Untersuchungszeitraumes konnten die Erweiterung des Repertories durch Aufspaltung und Reifung sowie ein möglicher Verlust von Rufklassen beobachtet werden.

---

Juvenile development of vocalizations in Greylag Geese (Anser anser)

Summary The juvenile vocal development of six Greylag Geese from hatching up to the age of 100 days was analysed by using sonagraphic methods. The calls were classified by means of the acoustic phenomenon, sonagrams, and context criteria. The juvenile repertoire included four main call types. During ontogeny, changes of call parameters mainly referred to phonetic characteristics, whereas syntactic parameters remained more or less constant. The frequency pitch of calls changed to higher levels during the first week of development while in older goslings this parameter decreased constantly. This held true for each individual and call type. Breaking of the voice, defined as an increment of noisy parts in the vocalizations, is described as a continuous long term process. Structural individuality of calls apparently was not based on one single parameter, but on the combination of several ones. Sex differences were found in the pitch of vocalizations, correlating with different body weights of male and female geese. Additional developmental processes of the vocal repertoire included differentiation, maturation of new call types, and apparent suppression of juvenile ones. The functional organization of the juvenile call system in Greylags is discussed.

---

---

Herrn Dr. Hans Löhrl zum 75. Geburtstag gewidmet     

Seasonal changes in the effects of arginine vasotocin and stretch on Anolis uterine contractions in vitro

We determined the effects of acute stretch on spontaneous and arginine vasotocin (AVT)-driven contractions of the Anolis carolinensis uterus in vitro. Whole uteri from reproductively inactive females (October) were placed in a bath of oxygenated 32 degrees C Anolis "Ringer's." Two initial tensions were utilized, 1.5 g or 15 g, the latter being an estimate of the tension on the wall of a uterine compartment. Uteri were then exposed to either saline or AVT (50 ng/ml), and spontaneous or AVT-driven contractions were recorded for 20 min with the use of a strain gauge and physiograph. A similar experiment was performed on uteri from reproductively active females in the summer (June). Our results indicate that the effects of acute stretch and AVT on uterine contractility were qualitatively similar in summer and fall. That is, AVT induced a tonic contraction; stretch decreased the duration of the tonic contraction; the saline-treated uteri exhibited spontaneous rhythmic contractions; AVT increased the amplitude of the rhythmic contractions, but only at the lower tension; there were no effects of AVT on the timing (contraction interval, duration, rest interval) of the rhythmic contractions; and stretch increased the frequency of the rhythmic contractions. Season greatly influenced the magnitude of these contractile phenomena. Uteri tested during the breeding season exhibited greater distensibility, an increase in the amplitude and duration of the AVT-driven tonic contraction, and an increase in the frequency of both spontaneous and AVT-driven rhythmic contractions because of a decrease in both contraction duration and rest interval.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunoelectron microscopical localization of phospholamban in adult canine ventricular muscle

The subcellular distribution of phospholamban in adult canine ventricular myocardial cells was determined by the indirect immunogold-labeling technique. The results presented suggest that phospholamban, like the Ca2+-ATPase, is uniformly distributed in the network sarcoplasmic reticulum but absent from the junctional portion of the junctional sarcoplasmic reticulum. Unlike the Ca2+-ATPase, but like cardiac calsequestrin, phospholamban also appears to be present in the corbular sarcoplasmic reticulum. Comparison of the relative distribution of phospholamban immunolabeling in the sarcoplasmic reticulum with that of the sarcolemma showed that the density of phospholamban in the network sarcoplasmic reticulum was approximately 35-fold higher than that of the cytoplasmic side of the sarcolemma, which in turn was found to be three- to fourfold higher than the density of the background labeling. However, a majority of the specific phospholamban labeling within 30 nm of the cytoplasmic side of the sarcolemma was clustered and present over the sarcoplasmic reticulum in the subsarcolemmal region of the myocardial cells, suggesting that phospholamban is confined to the junctional regions between the sarcolemma and the sarcoplasmic reticulum, but absent from the nonjunctional portion of the sarcolemma. Although the resolution of the immunogold-labeling technique used (60 nm) does not permit one to determine whether the specific labeling within 30 nm of the cytoplasmic side of the sarcolemma is associated with the sarcolemma and/or the junctional sarcoplasmic reticulum, it is likely that the low amount of labeling in this region represents phospholamban associated with sarcoplasmic reticulum. These results suggest that phospholamban is absent from the sarcolemma and confined to the sarcoplasmic reticulum in cardiac muscle.     

Escherichia coli mutants resistant to uncouplers of oxidative phosphorylation

Two mutant strains of Escherichia coli K 12 Doc-S resistant to the uncoupling agents 4,5,6,7-tetrachloro-2-trifluoromethyl benzimidazole and carbonyl cyanide m-chlorophenylhydrazone were isolated. These strains, designated TUV and CUV, were capable of (a) growth, (b) the transport of succinate and L-proline and (c) electron-transport-linked oxidative synthesis of ATP in the presence of titres of uncoupler which inhibited these processes in strain Doc-S. The inhibition of transport of L-proline by a fixed titre of uncoupler was sharply pH dependent in strain Doc-S: uptake was unaffected at pH 7.6 but completely inhibited at pH 5.6. This pH dependence was not shown by the resistant strains. We believe that uncouplers were equally accessible to their site(s) of action in the energy-conserving membrane of the sensitive and resistant strains. We conclude that uncoupler resistance in these strains of E. coli has arisen as a consequence of mutations which directly affect a specific site of uncoupler action within the cytoplasmic membrane, rather than as a consequence of a decrease in the permeability of cells to uncoupler.     

2-Phenylethylamine catabolism by Escherichia coli K12

Escherichia coli K12 grows on 2-phenylethylamine as sole carbon and energy source by converting it, via phenylacetaldehyde, to phenylacetic acid. Phenylacetaldehyde was formed by the action of an inducible amine oxidase and catalase activity was increased sixfold, presumably to ensure removal of the H2O2 that was expected to be a product of the amine oxidation. The phenylacetaldehyde was oxidized to phenylacetic acid by an inducible NAD+-dependent dehydrogenase. Mutants defective in phenylacetaldehyde dehydrogenase cannot grow on 2-phenylethylamine as carbon and energy source but can still use it as a nitrogen source.     

Conservation of δ-crystallin gene structure between ducks and chickens

A cloned chicken delta-crystallin cDNA was used to identify two putative delta-crystallin genes in the duck by Southern blot hybridization. A DNA fragment containing most of one of these genes was isolated from a library made in bacteriophage lambda Charon 28A containing genomic DNA from 14-day-old embryonic ducks. Electron microscopy, partial gene sequencing, primer extension analysis using duck mRNA, and comparison with the well-characterized chicken delta-crystallin genes suggest that our cloned duck delta-crystallin gene, like the chicken delta-crystallin genes, is 8-10 kb long and contains 17 exons. Hybridization and sequencing data show great similarity between the homologous 5' untranslated and coding exons of the duck and chicken delta-crystallin genes. Overall, the homologous introns also appear to have approximately 30% sequence similarity, and have been subject to deletion/insertion events. Our partial characterization of duck delta-crystallin gene sequences suggests that this avian and reptilian crystallin family has been conserved during evolution, as have the other crystallin gene families that are expressed in the eye lens.     

Animal model of acute pericarditis and its progression to pericardial fibrosis and adhesions: ultrastructural studies

To study the evolution of pericardial inflammation, we have developed a model of pericarditis in sheep by surgically injecting heat-killed staphylococci and Freund's adjuvant into the pericardial cavity under sterile conditions. The pericarditis evolved through the following phases: 1) inflammatory response, 2) mesothelial cell injury and desquamation, and 3) fibrotic phase. At 3-24 hr there was increased microvascular permeability, which resulted in the exudation of fluid, neutrophils, macrophages, and fibrin into the pericardial cavity and the pericardial interstitium. By 72 hr, large numbers of inflammatory cells were aggregated on the mesothelial surfaces and dispersed throughout the pericardial cavity, either as free-floating cells or located between strands of fibrin. At 6 days, fibrinolysis was apparent along the mesothelial surfaces; and newly formed collagen fibrils were deposited throughout the interstitial spaces and among the aggregated cells. These fibrils provided a matrix for the growth of new blood and lymphatic vessels into new connective tissue on both parietal and visceral pericardial surfaces. At 2 weeks, intrapericardial fibrosis had produced focal adhesions between the pericardial surfaces. At 1 month, extensive areas of the pericardial cavity were obliterated. By 9 months, there was a marked reduction in the numbers of cells and blood vessels and increased deposition of collagen and elastic fibers. The intrapericardial injection of heat-killed staphylococci and adjuvant provides a reproducible animal model to study the time course of pericardial inflammation.