Neurophysin and the neurosecretory fibres of the sheep infundibulum

Summary A cross-species reactive antiserum was used to study the cellular localization of neurophysin in the sheep hypothalamus and neurohypophysis. The immunofluorescent technique was used to follow the neurosecretory fibres emanating from the magnocellular nuclei to the lower infundibular stem. It was confirmed that neurophysin was present in both the internal and external zone of the infundibulum.This work was supported by a research grant from the New Zealand Medical Research Council.     

Differences between newly formed and recycled free small ribosome subunits in liver cytoplasm.

1. Ferritin has been isolated from the serum of four patients with iron overload by using two methods. 2. In method A, the serum was adjusted to pH 4.8 and heated to 70 degrees C. After removal of denatured protein, ferritin was concentrated and further purified by ion-exchange chromatography and gel filtration. In most cases, only a partial purification was achieved. 3. In method B, ferritin was extracted from the serum with a column of immuno-adsorbent [anti-(human ferritin)] and released from the column with 3M-KSCN. Further purification was achieved by anion-exchange chromatography followed by the removal of remaining contaminating serum proteins by means of a second immunoadsorbent. Purifications of up to 31 000-fold were achieved, and the homogeneity of the final preparations was demonstrated by polyacrylamide-gel electrophoresis. 4. Serum ferritin purified by either method has the same elution volume as human spleen ferritin on gel filtration on Sephadex G-200. Serum ferritin has a relatively low iron content and iron/protein ratios of 0.023 and 0.067 (mug of Fe/mug of protein) were found in two pure preparations. On anion-exchange chromatography serum ferritin has a low affinity for the column when compared with various tissue ferritins. Isoelectric focusing has demonstrated the presence of a high proportion of isoferritins of relatively high pI. 5. Possible mechanisms for the release of ferritin into the circulation are briefly discussed.     

Physiological properties of newly formed synapses between sympathetic preganglionic neurons and sympathetic ganglion neurons.

We have examined the physiological properties of transmission at newly formed synapses between sympathetic preganglionic neurons and sympathetic ganglion neurons in vitro. Chick neurons were labeled with fluorescent carbocyanine dyes before they were placed into culture (Honig and Hume, 1986), and were studied by making intracellular recordings during the first 2 weeks of coculture. Evoked monosynaptic excitatory postsynaptic potentials (EPSPs) were not observed until 48 h of coculture. Beyond this time, the frequency with which connected pairs could be found did not vary greatly with time. With repetitive stimulation, the evoked monosynaptic EPSPs fluctuated in amplitude from trial to trial and showed depression at frequencies as low as 1 Hz. To gain further information about the quantitative properties of transmission at newly formed synapses, we analyzed the pattern of fluctuations of delayed release EPSPs. In mature systems, delayed release EPSPs are known to represent responses to single quanta, or to the synchronous release of a small number of quanta. For more than half of the connections we studied, the histograms of delayed release EPSPs were extremely broad. This result suggested that either quantal responses are drawn from a continuous distribution that has a large coefficient of variation or that there are several distinct size classes of quantal responses. The pattern of fluctuations of monosynaptic EPSPs was consistent with both of these possibilities, and was inconsistent with the possibility that monosynaptic EPSPs are composed of quantal subunits with very little intrinsic variation. Although variation in the size of responses to single quanta might arise in a number of ways, one attractive explanation for our results is that the density and type of acetylcholine receptors varies among the different synaptic sites on the surface of developing sympathetic ganglion neurons.     

Accumulation of acetylcholinesterase at newly formed nerve--muscle synapases.

The mechanism of fluid transport in the developing preimplantation mouse embryo has been studiedin vitro by inhibiting zonular tight junction formation. Compaction, the morphogenetic process permitting zonular blastomere adhesions at the 8-cell stage, was suppressed by lowering extracellular calcium (Ca). The Ca threshold required for compaction is 0.04–0.06 mM, and in concentrations above the threshold, the rate of compaction is concentration dependent, whereas the rate of blastocyst formation is not and proceeds normally. At 0.02 mM Ca, both compaction and blastocyst development are completely prevented. Although focal tight and gap junctions are present, zonular tight junctions do not develop. We conclude that Ca is required for the maximization of cell-cell contact, but not for focal tight junction and gap junction formation. When early morulae are cultured in 0.02 mM Ca, small trophoblastic vesicles develop frequently with intracellular fluid vacuoles. If early 8-cell embryos are similarly cultured, cell division continues and many blastomeres acquire small intracellular membrane-bounded vaculoes. These coalesce, the cell volume increases, and the nucleus becomes eccentrically positioned, resulting in a giant vacuolated blastomere reminiscent of a miniaturized blastocyst. We propose that (1) vacuole formation may be an exaggeration of an intermediate intracellular step in fluid transport and (2) normal cell polarity established by zonular tight junctions is required for transcellular fluid transport.     

Characteristics of rapidly formed hydrogen-producing granules and biofilms

The physicochemical and microbiological characteristics of rapidly formed hydrogen-producing granules and biofilms were evaluated in the present study. Microbial species composition was examined using the 16S rDNA-based separation and sequencing techniques, and spatial distribution and internal structure of microbial components were evaluated by examining the confocal laser scanning microscope (CLSM) images. Phylogenetic analysis indicated that a pure culture of Clostridium pasteurianum-like bacterium (98% similarity) was found in microbial community of granules and biofilms. It is postulated that containing such a species favored the rapid immobilization of hydrogen-producing culture. Manure granules and biofilms secreted 24-35 mg extracellulous proteins and 142-175 mg extracellulous polysaccharides in each gram of culture (in VSS). Such a high productivity of extracellulous polymers (ECP), a bio-glue to facilitate cell-to-cell and/or cell-to-substratum interaction, may work as the driving forces for the immobilization of C. pasteurianum. As abundant proteins were noted in the granule cores, it can be derived that rapid formation of the hydrogen-producing granules could be due to the establishment of precursor protein-rich microbial nuclei.     

Neurosecretion. XII. The formation of neurosecretory granules in the earthworm,Lumbricus terrestris L.

Summary The granules of neurosecretory cells in the supraesophageal ganglion of the earthworm, Lumbricus terrestris L., are formed by the Golgi apparatus. The process of neurosecretion is discussed in relation to this observation.This research was aided by grants (B-840, B-2145 and 2M-6418) of the United States Public Health Service.     

Isolation and characterization of neurosecretory granules of the rat posterior pituitary gland

Summary Neurosecretory granules (NSG) of rat posterior pituitary glands were prepared by differential centrifugation techniques mainly according to the procedure as described by Barer, Heller and Lederis (1963). As revealed by electron microscopy, the recovery of neurophysin and the contents of enzymes, purified NSG were obtained in a pellet at 30 000 g/60 min (0.44 M sucrose). Eighteen h after injection of (³⁵S) cysteine into the supraoptic nucleus 60% of the recovered radioactivity in the neural lobe was found in the NSG, whereas 20% was found in the final supernatant (100 000 g/120 min). Sixteen days after injection the NSG and the final supernatant fraction contained fairly equal amount of (³⁵S) cysteine (approximately 40%). It is suggested that after a period of intragranular maturation neurophysin is extruded into an extragranular pool of neurosecretory material.With the use of conventional polyacrylamide-gel electrophoresis it was shown that the predominating proportion of radioactivity in the NSG after a hypothalamic injection of (³⁵S) cysteine was located within the neurophysin fraction A and in fraction B. Fraction B is suggested to be partly bound to the NSG membranes. When the NSG soluble and NSG insoluble proteins, obtained after lysis of NSG, were separated on polyacrylamide gels in the presence of sodium dodecylsulphate, the highly radioactive soluble protein was shown to consist of two components with average molecular weights of 12 300 and 14 600. Most of the proteins in the lysate were found in the NSG membranes, though less radioactive. A component with a mol.wt. of 37 000 was enriched in the membrane fraction. At longer times after isotope injection the high mol.wt. proteins, particularly those of the NSG membranes, contained increased amounts of radioactivity.Abbreviations NSG neurosecretory granule - NSM neurosecretory material - SON supraoptic nucleus The present investigation was supported by grants from Svenska Livförsäkringsbolags nämnd för medicinsk forskning from Svenska Sällskapet för Medicinsk forskning, from the Medical Faculty, University of Göteborg, and from the Swedish Medical Research Council (B 72-12X-2543-04A).We are indebted to Mrs. Marie-Louise Eskilsson, Mrs. Wally Holmberg and Mrs. Ulla Svedin for technical assistance, and to Miss Gull Grönstedt for careful secreterial work.     

Physiological properties of newly formed synapses between sympathetic preganglionic neurons and sympathetic ganglion neurons

We have examined the physiological properties of transmission at newly formed synapses between sympathetic preganglionic neurons and sympathetic ganglion neurons in vitro. Chick neurons were labeled with fluorescent carbocyanine dyes before they were placed into culture (Honig and Hume, 1986), and were studied by making intracellular recordings during the first 2 weeks of coculture. Evoked monosynaptic excitatory postsynaptic potentials (EPSPs) were not observed until 48 h of coculture. Beyond this time, the frequency with which connected pairs could be found did not vary greatly with time. With repetitive stimulation, the evoked monosynaptic EPSPs fluctuated in amplitude from trial to trial and showed depression at frequencies as low as 1 Hz. To gain further information about the quantitative properties of transmission at newly formed synapses, we analyzed the pattern of fluctuations of delayed release EPSPs. In mature systems, delayed release EPSPs are known to represent responses to single quanta, or to the synchronous release of a small number of quanta. For more than half of the connections we studied, the histograms of delayed release EPSPs were extremely broad. This result suggested that either quantal reponses are drawn from a continuous distribution that has a large coefficient of variation or that there are several distinct size classes of quantal responses. The pattern of fluctuation of monosynaptic EPSPs was consistent with both of these possibilities, and was inconsistent with the possibility that monosynaptic EPSPs are composed of quantal subunits with very little intrinsic variation. Although variation in the size of responses to single quanta might arise in a number of ways, one attractive explanation for our results is that the density and type of acetylcholine receptors varies among the different synaptic sites on the surface of developing sympathetic ganglion neurons.     

Fluorescent granules in the guinea-pig hypothalamus and their possible relation to neurosecretory substance

Summary Sections of guinea-pig hypothalamus stained by various methods have been studied under bright-field and fluorescence microscopy to determine the characteristics of refractile granules originally observed in fixed tissue stained for cholinesterases but also seen in unfixed, unstained material.When sections of fresh tissue or tissue fixed in formaldehyde: Na₂SO₄ were examined by fluorescence microscopy, the granules emitted with a wave-length between 600 and 620 m. The density and distribution of the granules closely paralleled that of one of the components that stained with the aldehyde-fuchsin and chrome-alum techniques for neurosecretory substance; granules were absent, however, from the area characterized by Herring substance, and from the stained perikarya and beaded axons of the supraoptic and paraventricular nuclei.The granules were most numerous in the median eminence and infundibular nuclei. Their concentration was about average in mature males and in lactating and hypophysectomized females; they were more abundant in late pregnancy and after ovariectomy and were particularly plentiful some weeks after hysterectomy. They were virtually absent from immature males and females.The possible relation of the granules to releasing factors or to vasotocin-like neurosecretory material is discussed.The authors are most grateful to Dr. J. S. Perry who carried out the surgery involved in these experiments. They also wish to express their thanks to Dr. R. B. Heap who, with Dr. Perry, gave invaluable help in planning the experiments and assessing the data; to Mr. S.P. Mann and Dr. D. F. Sharman for advice on the fluorescence technique and to Miss M. Hamon for skilled technical assistance.Publication of the colour pictures was made possible by a generous grant from the Wellcome Foundation; this support is gratefully acknowledged.Thanks are also due to the University of Alberta for a contribution towards the cost of reprints.     

The isolation of neurophysin-I and -II from bovine pituitary neurosecretory granules separated on a large scale from other subcellular organelles. Demonstration of slow equilibration of neurosecretory granules during centrifugation in a sucrose density gradient

1. An improved procedure for the isolation of neurosecretory granules from the posterior lobe of the bovine pituitary gland is described. 2. Of the total oxytocic and pressor activities present in the original tissue 80% was sedimentable. 3. The granules were separated from mitochondria by prolonged centrifugation in a sucrose density gradient. During a sedimentation period of 5hr. the granules moved progressively into denser regions of the gradient and the mitochondria remained at the top. 4. The biological activities of the granules were measured: the oxytocic activity was 11·56±1·63 and the pressor activity was 15·60±3·91 units/mg. of protein. 5. A protein was isolated from a lysate of granules prepared from 40 pituitary glands. Amino acid analysis showed that it consisted of a mixture of neurophysin-I and neurophysin-II in equal proportions. It accounted for 60% of the soluble granule protein and for 50% of the total granule protein. 6. The neurophysins present in the granules are associated with 19·1 units of oxytocic and 21·1 units of pressor activity/mg. of protein. 7. Starch-gel electrophoresis revealed the presence of both neurophysins in extracts of 15 pituitary glands studied individually. 8. We conclude that the polypeptide hormones, oxytocin and [8-arginine]-vasopressin, are normally closely associated with the two neurophysins within neurosecretory granules of the pituitary gland.     

Detection of aldehydefuchsin-positive neurosecretory granules in the cells of the suprachiasmatic nucleus of the rat.

Application of dark field microscopy to sections fixed with picric acid--formalin and stained with crotonaldehydefuchsin allows the demonstration of neurosecretory granules in the neurones of the suprachiasmatic nuclei of normal rats.     

Bilirubin production and conjugation from newly formed heme in isolated rat hepatocytes.

1. Heme synthesis from delta-aminolevulinic acid (delta-ALA) in freshly isolated rat hepatocytes was maximal at 100 microM with a rate of approx. 7 nmol being synthesized per g wet weight cells. 2. Approximately 8% of synthesized heme was converted to bilirubin and 50% of the newly synthesized bilirubin was conjugated. 3. The ratio of di to monoconjugate was approx. 2.5. Incorporation of delta-ALA into bilirubin was increased by additional delta-ALA, heme and was also doubled in cells isolated from animals treated with CoCl2. 4. Bilirubin formation was inhibited approx. 90% by in vitro treatment with heme oxygenase inhibitors zinc and tin protoporphyrin.     

Depletion of neurosecretory granules and membrane retrieval in the sinus gland of the crab

Summary Ultrastructural aspects of hormone release from the sinus gland of the crab Carcinus maenas, have been studied by incubation of glands in vitro (i) in high potassium-containing media to induce hormone release; (ii) in a high potassium-containing calcium-free medium in which depolarisation but no hormone release would be expected; and (iii) in control saline. Uptake of horseradish peroxidase into subcellular organelles was also studied.Many neurosecretory granules could be found in the nerve terminals but, in contrast to mammalian neurosecretory systems, structures resembling microvesicles were extremely scarce. High potassium stimulation in the presence of calcium caused an 18 % loss of granules from the nerve terminals associated with images of single and multiple exocytosis. It further caused an increase in vacuoles which could have accounted for 33 % of the membrane of the granules exocytosed. After incubation in high potassium-containing, calcium-free media there was no evidence either of exocytosis of granules or of an increase in the vacuole population. The population of sparse microvesicle-like structures was not significantly altered by incubation in either high potassium medium. Horseradish peroxidase reaction product could be found only in vacuoles of tissues stimulated by high potassium concentrations in the presence of calcium. It is concluded that this depolarising stimulus produces, in the presence of calcium, the release by exocytosis of about one sixth of all the granules in the sinus gland, and that vacuoles are the organelle responsible for the recapture of membrane after the exocytosis.     

Difference in distribution of membrane proteins between low- and high-density secretory granules in parotid acinar cells

Secretory granules (SGs) are considered to be generated as immature granules and to mature by condensation of their contents. In this study, SGs of parotid gland were separated into low-, medium-, and high-density granule fractions by Percoll-density gradient centrifugation, since it was proposed that the density corresponds to the degree of maturation. The observation with electron microscopy showed that granules in the three fractions were very similar. The average diameter of high-density granules was a little but significantly larger than that of low-density granules. Although the three fractions contained amylase, suggesting that they are all SGs, distribution of membrane proteins was markedly different. Syntaxin6 and VAMP4 were localized in the low-density granule fraction, while VAMP2 was concentrated in the high-density granule fraction. Immunoprecipitation with anti-syntaxin6 antibody caused coprecipitation of VAMP2 from the medium-density granule fraction without solubilization, but not from Triton X-100-solubilized fraction, while VAMP4 was coprecipitated from both fractions. Therefore, VAMP2 is present on the same granules, but is separated from syntaxin6 and VAMP4, which are expected to be removed from immature granules. These results suggest that the medium-density granules are intermediates from low- to high-density granules, and that the membrane components of SGs dynamically change by budding and fusion during maturation.     

Translocation of pleckstrin requires its phosphorylation and newly formed ligands

Pleckstrin is the major substrate of protein kinase C (PKC) in platelets. We sought to determine whether pleckstrin phosphorylation is sufficient to target the soluble protein to binding sites. Permeabilization of platelets by streptolysin O (SLO) was used to separate bound and soluble pleckstrin. Platelets were incubated with phorbol 12-myristate 13-acetate (PMA) and/or guanosine 5'-[gamma-thio]triphosphate (GTP[S]) in the presence of [gamma-(32)P]ATP and SLO. PMA stimulated pleckstrin phosphorylation, but this pleckstrin diffused from permeabilized platelets. Addition of GTP[S] with PMA caused up to 40-50% of pleckstrin to be retained within platelets and enhanced secretion of platelet 5-hydroxytryptamine. PKC alpha pseudosubstrate peptide inhibited pleckstrin phosphorylation, the binding of pleckstrin and secretion. After extraction of permeabilized platelets containing bound pleckstrin with Triton X-100, the protein was solubilized. Thus, phosphorylated pleckstrin was retained in platelets only after activation of GTP-binding proteins that stimulate the formation of membrane-bound pleckstrin ligands. Translocation of pleckstrin may facilitate the associated secretion.     

Relationships between vegetation zonation and environmental factors in newly formed tidal marshes of the Yangtze River estuary

The Yangtze River delta is characterized by rapidly accreting sediments that form tidal flats that are quickly colonized by emergent vegetation including Scirpus mariqueter and the invasive species Spartina alterniflora. We measured soil surface elevation, water table depth, soil salinity, water content and compaction in the tidal flat, the Scirpus and Spartina zones and their borders to identify relationships between environmental factors and colonization by Scirpus and Spartina. With increasing elevation from tidal flat to Spartina, inundation frequency and duration, moisture and depth to water table decreased whereas soil salinity, temperature and compaction increased. High soil moisture and groundwater and low salinity were the characteristics of the tidal flat and its border with Scirpus. The Spartina zone and its border with Scirpus were characterized by greater salinity and elevation relative to the other zones. Our findings suggest that soil salinity controls patterns of plant zonation in the newly formed tidal salt marshes whereas elevation is of secondary importance. Our results suggest that patterns of vegetation zonation in tidal marshes of the Yangtze River delta are controlled by environmental factors, especially (low) salinity that favors colonization by Scirpus in the lower elevations of the marsh.     

Morphological heterogeneity of secretory granules of rat Clara cells: an immunocytochemical study.

The secretory granules of rat bronchiolar Clara cells were classified into different types by their ultrastructural appearances followed by immunocytochemistry using anti-rat 10 kDa Clara cell-specific protein (10 kDa CCSP) antibody. One predominant type was the oval to round granule (type A granule), of which the matrix was composed of a map-like mixture of electron-dense and less electron-dense material. Another predominant type was the rod-shaped granule (type B granule). The content of type B granules varied from a finely fibrillar (type B1 granule) to an electron-dense, rod-like (type B3 granule) structure. Various intermediate types (type B2 granule) between type B1 and B3 granules were also found. Small cytoplasmic vesicles were found occasionally in close proximity to type B2 or B3 granule. Another type of granule (type C granule) was large, up to 8 microns in diameter, and contained a moderately electron-dense amorphous matrix. Both type A and C granules stained at a similar density with the antibody. The nascent form of type A granules, which was found in the vicinity to the trans face of the Golgi apparatus, was also labeled. On the other hand, the labeling density of type B granules varied: type B1 granules were almost devoid of immunolabeling, whereas type B3 granules were intensely labeled. Type B2 granules stained with the antibody; however, the labeling density was less than that of type B3 granules. The small cytoplasmic vesicles of type B2 granules were labeled.(ABSTRACT TRUNCATED AT 250 WORDS)     

Common antigens of mouse oval and biliary epithelial cells. Expression on newly formed hepatocytes

Two antigens - A6 and G7 - shared by mouse biliary epithelial and oval cells were revealed by monoclonal antibodies raised in rat immunized with oval-cell-enriched liver fraction. Oval cells were induced in CBA or F1 (CBA x C57BL6) mice by a combination of a single injection of the alkylating drug Dipin with partial hepatectomy. In normal liver A6 antigen was localized, using light and electron microscopy, in biliary epithelial cells of all ducts including Hering canals. Some bile ductal and Hering cells were A6-negative. Occasionally, A6 antigen was present in single hepatocytes forming the periportal ends of hepatic cords. In preneoplastic and tumorous liver A6 antigen was present in bile ductal and oval cells and in a fraction of newly formed hepatocytes and tumor cells. G7 antigen was revealed in normal, precancerous and tumorous liver in biliary epithelial and oval cells but not in hepatocytes. A6 and G7 antigens were not liver-specific: they were expressed in various normal organs and tissues, especially in epithelia. In studies of mouse liver lineages A6 antigen can be used as a common marker of biliary epithelial and oval cells and hepatocytes at certain stages of differentiation. G7 antigen is a marker of oval and biliary epithelial cells. There was a striking similarity in A6 antigen localization to that of human blood group antigens in normal liver and liver tumors. A6 antigen may thus provide a useful tool for the study of neoexpression of human blood group antigens in liver tumors.