Neurophysin heterogeneity. Difference between newly formed and aged neurosecretory granules

Neurosecretory granules from bovine neurohypophyses were isolated on iso-osmotic gradients. The content of the granules was analyzed by analytical and two-dimensional gel electrophoreses. The distributions in the gels of vasopressin precursor and neurophysins were detected by radioimmunoassays. Analytical gel electrophoresis of the content of a crude granule preparation showed the presence of different populations of neurophysin molecules. Further analysis demonstrated that vasopressin-neurophysin and oxytocin-neurophysin can be subdivided into molecules with different pI values. Whereas newly formed granules showed two main spots of neurophysin with pI of 5.0 and 5.6, aged granules contain in addition to these different populations of neurophysin-like material, some of which had a basic pI. Vasopressin precursor activity was detected in spots containing proteins with acidic pI and Mr approximately 18,000 and also in proteins of Mr = 8,000-10,000 migrating in the basic region of the gel. The results suggest that in the neural lobe there is an aging process which gives rise to several subpopulations of neurophysins. The different forms of vasopressin-associated bovine neurophysin and oxytocin-associated bovine neurophysin are only found in the granules which are not required for release.     

Man's impact on a newly formed reservoir

A newly formed reservoir in the southwestern part of the United States was analyzed for man's impact on the eutrophication of the impoundment. The analysis of the ¹⁴C net productivity (mg ¹²C/m² per day) indicated that the area studied was naturally eutrophic. Significant differences in net production were observed among the sites, as the area where man's recreational activities are highly concentrated had a significantly higher production rate than the other sites investigated. Mean monthly estimate of production for all the sites, and monthly and yearly estimates for the area studied are also included.     

Retrotransposon-mediated restoration of Chlorella telomeres: accumulation of Zepp retrotransposons at termini of newly formed minichromosomes

To elucidate the contribution of LINE-like retrotransposon Zepp elements to the formation and maintenance of chromosomal telomeres, newly formed minichromosomes in irradiated Chlorella vulgaris cells were isolated and structurally characterized. A minichromosome (miniV4) of ~700 kb in size contained a Zepp cluster taking the place of the telomeric repeats on one terminus, whereas the other end of this chromosome consisted of canonical telomeric repeats. The Zepp copies in this cluster were in a tandem array with their poly(A) tails towards the centromere. Another minichromosome Y32 (~400 kb in size) was shown to have several copies of Zepp elements on both termini. On the right arm terminus, two copies of Zepp were found in a tandem array with poly(A) tracts facing towards the chromosomal end. The poly(A) tail and the 3′-end of ~400 bp of the distal copy were replaced by the telomeric repeats. On the 5′-side of the proximal copy was another Zepp element in the reverse orientation. These newly formed telomeric structures are very similar to those previously found in the left arm of chromosome I and the terminus of an unidentified chromosome and support the model of Zepp-mediated restoration and maintenance of Chlorella telomeres.     

Junctional form of acetylcholinesterase restored at nerve-free endplates.

Rat soleus muscles were ectopically innervated by implanting a foreign nerve in an endplate-free region of muscle and, 2–3 weeks later, cutting the original nerve. The junctional, 16 S form of acetylcholinesterase (AChE) and focal staining for AChE disappeared from the old endplate region within a few days after denervation. In muscles with an ectopic nerve, but not in paired control muscles, 16 S AChE and focal staining were restored in the old endplate region 1–2 weeks after denervation even though nerve fibers could not be detected in that region. These results suggest that the nerve exerts a local effect, specifying the site at which junctional AChE appears, and a nonlocal effect, perhaps mediated by muscle activity, regulating the amount of junctional AChE.     

Incorporation of newly formed lecithin into peripheral nerve myelin

Radioactive choline was used to study the metabolism and movement of choline-containing phospholipids in peripheral nerve myelin of adult mice. Incorporation at various times after intraperitoneal injection was measured in serial segments of sciatic nerve as well as in myelin isolated from those segments. At no time (1 h to 35 days) could a proximal-distal difference in the extent of labeling be demonstrated. This finding suggests that incorporation of precursor choline phospholipids into nerve membranes is a local event, with little contribution from the neuronal perikaryon via axoplasmic transport. Autoradiographic investigations were undertaken to elucidate the pattern of movement of radioactive choline-labeled phospholipids, predominantly lecithin, into the myelin sheaths of the sciatic nerve. A sequence of autoradiographs was prepared from animals sacrificed between 20 min and 35 days after a microinjection of precursor directly into the nerve. Analysis of these autoradiograms revealed that labeling is initially concentrated in the Schwann cell cytoplasm. Later, the label moves first into the outer regions of the myelin sheaths and is eventually distributed evenly throughout the inner and outer layers of the sheath. At no time is there a build-up of label in the axon. The rate of uptake of precursor and subsequent redistribution of lecithin into the myelin were also examined in frog sciatic nerve (18 degrees C). Both uptake and redistribution processes were considerably slower in the cold-blooded animal.     

Distribution of newly formed fatty acids among glycerolipids of isolated perfused rat liver.

Livers of chow fed rats were perfused 1-3 h with buffer, glucose, albumin, and red blood cells, made up in 100 percent D(2)O. Glycerolipids were isolated and the deuterated fatty acids determined by gas chromatography with mass spectrometry on Silar 5 CP. Percentage of replacement by deuterated acids ranged from 1 to 14, of which palmitate was 87 percent. Differences were found in total lipid class and in subcellular distribution of the newly synthesized acids. Microsomes had 37 percent more deuterated acids than the total or floating fat. At 3 h the highest replacement was found in diacylglycerols (17 percent) and free fatty acids (11 percent). Of the palmitate in hepatic choline and ethanolamine phosphatides, 6.9 percent and 4.7 percent, respectively, contained dueterium. The serine and inositol phosphatides had a higher proportion of deuterated palmitate (7.7 percent) than other phosphatides. The data support the hypothesis that palmitate is incorporated into glycerolipids largely via de novo synthesis while stearate enters them by deacylation-acyl transfer replacement.     

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.     

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.     

Distribution of newly formed ribosomal proteins in HeLa cell fractions

The distribution of newly formed ribosomal proteins between cytoplasmic, nucleoplasmic, and nucleolar fractions of HeLa cells was determined. All but a few of the newly formed ribosomal proteins were concentrated 10- to 50-fold in the nucleolus and two- to fivefold in the nucleoplasm. Nevertheless, substantial amounts were found in the cytoplasm. Pretreatment of cells with actinomycin D to deplete the nucleolar pool of ribosomal precursor RNA had no effect on the concentration of newly formed ribosomal proteins in the nucleus, but did lead to an increased amount in the nucleoplasm at the expense of the nucleolus.     

Mobilization of Stowaway-like MITEs in newly formed allohexaploid wheat species

Transposable elements (TEs) dominate the genetic capacity of most eukaryotes, especially plants, where they can account for up to 90?% of the genome, such as in wheat. The relationship between TEs and their hosts and the role of TEs in organismal biology are poorly understood. In this study, we have applied next generation sequencing, together with a transposon display technique in order to test whether a Stowaway-like MITE, termed Minos, transposes following allopolyploidization events in wheat. We have generated a 454-pyrosequencing database of Minos-specific amplicons (transposon display products) from a newly formed wheat allohexaploid and its parental lines and retrieved hundreds of novel MITE insertions in the allohexaploid. Clear mobilization of Minos was also seen by site-specific PCR analysis and sequence validation. In addition, using real-time qPCR analysis we observed an insignificant change in the relative quantity of Minos from the expected value of merging the two parental genomes, indicating that, despite its activation, no significant burst in Minos copy number can be seen in the newly formed allohexaploid. Interestingly, we found that CCGG sites surrounding Minos underwent massive hypermethylation following the allohexaploidization process. Our data suggest that MITEs have maintained their capacity for activity throughout the evolution of wheat and might be epigenetically deregulated in the first generations following allopolyploidization.     

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.     

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.     

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.     

游泳动物对长江口新生盐沼湿地潮沟生境的利用

利用长袋网(fyke net)采集了长江口新生盐沼湿地潮沟内的游泳动物,分析了该类盐沼湿地的鱼类栖息地利用。结果表明,3次调查共记录到游泳动物20种,其中鱼类15种,虾蟹类5种。生态类群主要以淡水性种类(10种)和河口性种类(6种)为主,洄游性种类(3种)和海洋性种类(1种)较少。从数量组成来看,游泳动物群落主要由安氏白虾(35.4%)、棕刺虾虎鱼(17.7%)、贝氏餐(17.7%)、长蛇鮈(9.2%)和日本沼虾(4.6%)等少数几个物种占优势。这些游泳动物大多为稚幼个体,表明长江口新生盐沼湿地是许多鱼类和甲壳动物的重要育幼场所。     

Protein conformation stabilized by newly formed turns for thermal resilience

Thermally stable or resilient proteins are usually stabilized at intermediate states during thermal stress to prevent irreversible denaturation. However, the mechanism by which their conformations are stabilized to resist high temperature remains elusive. Herein, we investigate the conformational and thermal stability of transforming growth factor-β1 (TGF-β1), a key signaling molecule in numerous biological pathways. We report that the TGF-β1 molecule is thermally resilient as it gradually denatures during thermal treatment when the temperature increases to 90°C–100°C but recovers native folding when the temperature decreases. Using this protein as a model, further studies show the maintenance of its bioactive functional properties after thermal stress, as demonstrated by differentiation induction of NIH/3T3 fibroblasts and human mesenchymal stem cells into myofibroblasts and chondrocytes, respectively. Molecular dynamic simulations revealed that although the protein’s secondary structure is unstable under thermal stress, its conformation is partially stabilized by newly formed turns. Given the importance and/or prevalence of TGF-β1 in biological processes, potential therapeutics, and the human diet, our findings encourage consideration of its thermostability for biomedical applications and nutrition.     

The acquisition of antigen cross-presentation function by newly formed dendritic cells

The development of Ag-presenting functions by murine dendritic cells (DCs) of the CD8(+) DC lineage was studied using a Flt-3 ligand stimulated bone-marrow culture system. Although newly formed DCs of this lineage are capable of Ag uptake and efficient presentation to T cells on MHC class II, they initially lack the ability to cross-present exogenous Ags on MHC class I. Cross-presentation capacity is acquired as a subsequent maturation step, promoted by cytokines such as GM-CSF. The development of cross-presentation capacity by the DCs in these cultures may be monitored by the parallel development of DC surface expression of CD103. However, the expression of CD103 and cross-presentation capacity are not always linked; therefore, CD103 is not an essential part of the cross-presentation machinery. These results explain the considerable variability in CD103 expression by CD8(+) DCs as well as the findings that not all DCs of this lineage are capable of cross-presentation.