Elektronenmikroskopische Untersuchungen am Nucleus praeopticus der Kröte (Bufo vulgaris formosus)

Zusammenfassung Die Feinstruktur der neurosekretorischen Nervenzellen des Nucleus praeopticus magnocellularis der Kröte (Bufo vulgaris formosus) und ihre Umgebung wurde untersucht.Die neurosekretorischen Zellen enthalten drei Arten von osmiophilen Gebilden: die neurosekretorischen Elementargranula, die neurosekretorischen Kügelchen und die Einschlußkörper.Die neurosekretorischen Elementargranula besitzen einen Durchmesser von 1000–3000 Å, durchschnittlich von 1300–1500 Å. Sie entstehen im Golgi-Apparat (Perikaryon) der betreffenden Zellen wie bei den schon beschriebenen anderen Tierarten.Die neurosekretorischen Kügelchen haben einen Durchmesser von 4000 Å bis zu mehreren . Sie kommen zuerst in den Ergastoplasmacisternen des Perikaryons vor und wandern dann innerhalb des Axons caudalwärts ab, ebenso wie die Elementargraunla, verlieren sich aber vor dem Erreichen der Neurohypophyse. Nach Lage und Gestalt entsprechen sie den Kolloidtropfen, die von vielen Lichtmikroskopikern für die neurosekretorischen Zellen niederer Vertebraten beschrieben wurden.Die Einschlußkörper treten vornehmlich im zentralen Bezirk des Perikaryons in Erscheinung. Sie sind so groß wie die Mitochondrien und besitzen verschiedene Innenstrukturen. Auf Grund der Struktur und der histochemischen Reaktion möchten wir diese Einschlußkörper den Lipofuscingranula mit saurer Phosphatase zuordnen.Die neurosekretorischen Nervenzellen schmiegen sich an den die Kapillare umgebenden Perivaskularraum unmittelbar an, innerhalb dessen die Basalmembran unvollkommen ausgebildet ist oder ganz fehlt.Stellenweise dehnt sich ein Abschnitt des Endothels durch den Perivaskularraum hindurch entlang der Außenfläche des Perivaskularraums aus, wobei sich die Endothelzellen der Kapillare und die neurosekretorischen Nervenzellen direkt berühren können. Eine poröse Bauweise des Endothels wurde nicht nachgewiesen. Zwischen den Ependymzellen des III. Ventrikels und den darunterliegenden neurosekretorischen Nervenzellen sind oftmals auffallend große Extrazellularräume zu beobachten, die durch den Spaltraum der benachbarten Ependymzellen mit dem Ventrikellumen kommunizieren. Sie enthalten mikrovilliartige Ausläufer der Ependymzellen und die geschilderten, neurosekretorische Bildungen führenden Axone. Eine Ausstoßung dieser Axone in den Ventrikel wurde nicht festgestellt.Diese Untersuchung wurde zum Teil mit finanzieller Unterstützung durch das Japanische Unterrichtsministerium im Jahre 1963 durchgeführt.Der kurze Inhalt dieser Arbeit wurde unter dem Thema 'Electron microscopic studies on the praeoptic nucleus in the toad am 5. und 6. September 1963 auf dem Kongreß für Endokrinologie in Gunma, Japan, vorgetragen.     

Distribution of sodium-plus-potassium-stimulated adenosine-triphosphatase activity in isolated nerve-ending particles

1. A rapid method for the isolation of nerve-ending particles from brain is described. This involved the centrifugation of the large-granule fraction over a discontinuous density gradient consisting of 3% (w/v) and 13% (w/v) Ficoll dissolved in 0.32m-sucrose. The results of the biochemical as well as morphological identification of nerve-ending particles are given. 2. Approx. 20% of the (Na(+)+K(+))-stimulated adenosine-triphosphatase activity originally present in the cerebral grey-matter suspension was recovered in the fraction consisting principally of large nerve-ending particles (approx. 1mu in diameter). The activity of the adenosine triphosphatase/mg. of protein in the nerve-ending fraction approximated to that in the small-granule fraction after the treatment with glycol ether diamine-tetra-acetic acid. The conclusion was drawn that the synaptic structure, supposedly the limiting membrane of the nerve-ending particle, is one of the feasible sites of localization of the (Na(+)+K(+))-stimulated adenosine-triphosphatase activity in cerebral tissues. Adenosine triphosphatase in purified cerebral mitochondria was not stimulated by Na(+). 3. No qualitative differences were found between the (Na(+)+K(+))-stimulated adenosine-triphosphatase activities exhibited by the nerve-ending particles and by the cerebral small-granule fraction with respect to pH-dependence, cation requirements and susceptibility to ouabain.     

Improved maintenance of adult rat hepatocytes in a new serum-free medium in the presence or absence of barbiturates

Summary For serum-free primary culture of adult rat hepatocytes, a synthetic medium DM-160 and rat-tail collagen were selected for the basal medium and for the culture substratum, respectively. Barbiturates, such as phenobarbital and 1-ethyl-5-isobutylbarbiturate, efficiently supported survival of hepatocytes and maintained their morphologic features at lower concentrations under the serum-free conditions than under the serum-supplemented conditions. However, the hepatocyte survival rates under the serum-free conditions were lower than those under the serum-supplemented conditions in the presence or absence of barbiturates. Supplementation of the basal medium with a combination of five groups of factors (5Fs), such as eight amino acids (Ala, Arg, Gly, Ile, Met, Phe, Pro, and Trp), two unsaturated fatty acids (linoleate and oleate), a protease inhibitor (aprotinin), three vitamins (A, C, and E), and five trace elements (Mn, Fe, Cu, Zn, and Se), improved the hepatocyte survival under the serum-free conditions in the presence or absence of barbiturates. In other words, the serum could be completely substituted by the 5Fs. Hepatocyte cultures maintained in the 5Fs-suppelemented basal medium showed excellent induction of tyrosine aminotransferase activity in response to dexamethasone in the presence or absence of barbiturates. The efficiency of the 5Fs-supplemented basal medium for maintaining hepatocytes was not inferior to those of other media in common use with hepatocytes, such as Williams' medium E and Waymouth's medium MB-752/1. In conclusion, maintenance of functional hepatocytes in serum-free primary culture could be improved by use of the new medium preparation (the 5Fs-supplemented DM-160) in the presence of barbiturates. This work was supported by a grant no. 61771923 from the Ministry of Education, Science and Culture of Japan.     

Production and characterization of recombinant human neutrophil chemotactic factor

A putative mature human neutrophil chemotactic factor (NCF) corresponding to the C-terminal 72 amino acids of its precursor was directly produced in Escherichia coli by recombinant DNA technology. Human NCF was present in both the soluble and insoluble protein fractions of the homogenate of host cells, and it was partially purified as a water-soluble polypeptide from both fractions, separately. The partially purified NCF preparation was highly purified to an endotoxin-free homogeneous polypeptide by means of CM-Sepharose CL-6B column chromatography and gel filtration on Toyopearl HW-55. No difference between the human NCF preparations purified from both starting materials could be found concerning purity, primary structure, solubility, molecular weight, and chemotactic activity for human neutrophils. The amino acid sequence of recombinant human NCF was identical to the sequence deduced from the cDNA sequence. A methionine residue due to the translation initiation codon was removed. Recombinant human NCF was found to be biologically active and to exhibit chemotactic activity for human neutrophils in vitro and cause a neutrophil infiltration in vivo in mice.     

Comparative study of the sugar chains of gamma-glutamyltranspeptidases purified from human hepatocellular carcinoma and from human liver

The sugar chains of gamma-glutamyltranspeptidases, purified from human hepatoma and from normal human liver, were released quantitatively as oligosaccharides by hydrazinolysis. Comparative study of their structures revealed that the following structural alterations are induced by hepatocyte carcinogenesis: 1) high mannose type sugar chains are detected in the hepatoma enzyme but not in the normal liver enzyme; 2) abnormal biantennary sugar chains containing C-2,4 outer chain branches newly appeared; 3) the total amounts of tri- and tetraantennary sugar chains containing C-2,6 outer chain branches increased up to three times.     

Recombinational hotspot specific to female meiosis in the mouse major histocompatibility complex

Thewm7 haplotype of the major histocompatibility complex (MHC), derived from the Japanese wild mouseMus musculus molossinus, enhances recombination specific to female meiosis in theK/A interval of the MHC. We have mapped crossover points of fifteen independent recombinants from genetic crosses of thewm7 and laboratory haplotypes. Most of them were confined to a short segment of approximately 1 kilobase (kb) of DNA between theA 3 andA 2 genes, indicating the presence of a female-specific recombinational hotspot. Its location overlaps with a sex-independent hotspot previously identified in theMus musculus castaneus CAS3 haplotype. We have cloned and sequenced DNA fragments surrounding the hotspot from thewm7 haplotype and the corresponding regions from the hotspot-negative B10.A and C57BL/10 strains. There is no significant difference between the sequences of these three strains, or between these and the published sequences of the CAS3 and C57BL/6 strains. However, a comparison of this A3/A2 hotspot with a previously characterized hotspot in theE gene revealed that they have a very similar molecular organization. Each hotspot consists of two elements, the consensus sequence of the mouse middle repetitive MT family and the tetrameric repeated sequences, which are separated by 1 kb of DNA.The nucleotide sequence data reported in this paper have been submitted to the DNA Data Bank of Japan nucleotide sequence database and have been assigned the accession numbers d90007-9. Offprint requests to: T. Shiroishi.     

Effects of the Histamine H3-Agonist (R)-α-Methylhistamine and the Antagonist Thioperamide on Histamine Metabolism in the Mouse and Rat Brain

To study the feedback control by histamine (HA) H3-receptors on the synthesis and release of HA at nerve endings in the brain, the effects of a potent and selective H3-agonist, (R)-alpha-methylhistamine, and an H3-antagonist, thioperamide, on the pargyline-induced accumulation of tele-methylhistamine (t-MH) in the brain of mice and rats were examined in vivo. (R)-alpha-Methylhistamine dihydrochloride (6.3 mg free base/kg, i.p.) and thioperamide (2 mg/kg, i.p.), respectively, significantly decreased and increased the steady-state t-MH level in the mouse brain, whereas these compounds produced no significant changes in the HA level. When administered to mice immediately after pargyline (65 mg/kg, i.p.), (R)-alpha-methylhistamine (3.2 mg/kg, i.p.) inhibited the pargyline-induced increase in the t-MH level almost completely during the first 2 h after treatment. Thioperamide (2 mg/kg, i.p.) enhanced the pargyline-induced t-MH accumulation by approximately 70% 1 and 2 h after treatment. Lower doses of (R)-alpha-methylhistamine (1.3 mg/kg) and thioperamide (1 mg/kg) induced significant changes in the pargyline-induced t-MH accumulation in the mouse brain. In the rat, (R)-alpha-methylhistamine (3.2 mg/kg, i.p.) and thioperamide (2 mg/kg, i.p.) also affected the pargyline-induced t-MH accumulation in eight brain regions and the effects were especially marked in the cerebral cortex and amygdala. These results indicate that these compounds have potent effects on HA turnover in vivo in the brain.     

Carbachol-Induced Cosecretion of Immunoreactive Atrial Natriuretic Peptides with Catecholamines from Cultured Bovine Adrenal Medullary Cells

The distribution and secretion of atrial natriuretic peptides (ANPs) were investigated in bovine adrenal medulla. (1) Cultured bovine adrenal medullary cells (2 x 10(6)/dish) contained 100.4 +/- 6.0 fmol of immunoreactive ANP (IR-ANP) and 207.3 +/- 6.6 nmol of catecholamines as epinephrine plus norepinephrine. (2) Stimulation of nicotinic but not muscarinic acetylcholine receptors caused a cosecretion of IR-ANP and catecholamines corresponding to the ratio of IR-ANP to catecholamines in cultured bovine adrenal medullary cells. (3) Carbachol-stimulated secretion of IR-ANP was dependent on the presence of extracellular Ca2+. (4) Chromaffin granules isolated from bovine adrenal medulla contained large amounts of IR-ANP and catecholamines, in the same ratio as did cultured adrenal medullary cells. (5) Reverse-phase HPLC analysis showed that both stored and secreted IR-ANP consisted of two components, which eluted at the position of ANP(99-126) or ANP(1-126). These results indicate that ANPs are stored as ANP(99-126) and ANP(1-126) in chromaffin granules, and are cosecreted in parallel with catecholamines in a Ca2+-dependent manner by the stimulation of nicotinic acetylcholine receptors.     

Endothelin receptors in human parathyroid gland.

We studied whether specific receptors for endothelins (ETs) exist in human parathyroid tissues and whether ETs may have any effect on secretion of PTH from parathyroid cells. Binding studies using [125I]ET-1 to the parathyroid membranes obtained from patients with hyperparathyroidism (2 adenomas, 2 hyperplasias) revealed that ET-1 competitively inhibited the binding of [125I]ET-1 to the membranes (the apparent Kd: 62 +/- 18 pM), whereas ET-3 showed biphasic and less steep inhibition curve than ET-1 in all tissue membranes examined. Northern blot analysis of poly(A)+ RNA from the parathyroid adenoma clearly demonstrated gene expression of both ETA and ETB receptors as well as preproET-1. ET-1 inhibited basal PTH secretion from dispersed adenoma cells more potently than ET-3. The present study clearly demonstrates the presence of both ETA and ETB receptor subtypes in human parathyroid tissues through which ETs may modulate PTH secretion in an autocrine and/or paracrine manner.