Human joining peptide: a proopiomelanocortin product secreted as a homodimer

The human (h) POMC gene sequence predicts a 30 amino acid joining peptide (JP) separating the N-terminal fragment [POMC(1-76) or hNT] and ACTH within their common precursor. We used an anti-serum directed against the amidated COOH-terminal end of mouse JP to develop a RIA for the predicted hJP molecule. Immunoreactive JP was detected in tissue extracts from human normal pituitary, ACTH-secreting pituitary- and nonpituitary tumors, and in plasma from patients with ACTH hypersecretory syndromes. Its molar concentration was of the same order of magnitude as, and correlated with, that of the other POMC peptides. Gel exclusion chromatography in 1% formic acid and 6 M guanidine-HCl revealed a predominant immunoreactive material with an apparent mol wt of ca. 6000. After reduction with dithiothreitol this material was recovered in an elution volume identical to that of purified hJP and corresponding to a mol wt of ca. 3000. These data show that POMC processing generates a COOH terminally amidated hJP predominantly secreted as a homodimer, probably through disulfide bonding between the single Cys9 residue of two molecules.     

A cell type-specific enhancer drives expression of the chick muscle acetylcholine receptor alpha-subunit gene

The regulation of acetylcholine receptor alpha-subunit gene expression was analyzed by transient expression assays. Using rabbit beta-globin cDNA as a reporter gene, we have confirmed that the 5'-flanking sequence of the chicken acetylcholine receptor alpha-subunit gene directs specific expression in differentiated C2C12 cells, a mouse muscle cell line, but not in undifferentiated C2C12 cells and mouse 3T3 fibroblasts. Testing chimeric plasmids containing Bal31 deletion mutants of the alpha-subunit gene upstream sequence, we found the -116 to -81 region of the alpha-subunit to be responsible for tissue- and stage-specific expression. This 36 bp fragment stimulates the activity of both alpha-subunit and SV40 promoters in a distance- and orientation-independent manner, thus fulfilling the criteria of an enhancer.     

Selenium-dependent glycine reductase: differences in physicochemical properties and biological activities of selenoprotein A components isolated from Clostridium sticklandii and Clostridium purinolyticum

The selenoprotein A component of the glycine reductase complex of Clostridium sticklandii was shown to differ in certain properties from the selenoprotein A produced by a purine-fermenting organism, Clostridium purinolyticum. Both proteins contain one selenocysteine and two cysteine residues.     

Ferricrocin functions as the main intracellular iron-storage compound in mycelia ofNeurospora crassa

Summary Neurospora crassa produces several structurally distinct siderophores: coprogen, ferricrocin, ferrichrome C and some minor unknown compounds. Under conditions of iron starvation, desferricoprogen is the major extracellular siderophore whereas desferriferricrocin and desferriferrichrome C are predominantly found intracellularly. Mössbauer spectroscopic analyses revealed that coprogen-bound iron is rapidly released after uptake in mycelia of the wild-typeN.crassa 74A. The major intracellular target of iron distribution is desferriferricrocin. No ferritin-like iron pools could be detected. Ferricrocin functions as the main intracellular iron-storage peptide in mycelia ofN. crassa. After uptake of ferricrocin in both the wild-typeN. crassa 74A and the siderophore-free mutantN. crassa arg-5 ota aga, surprisingly little metabolization (11%) could be observed. Since ferricrocin is the main iron-storage compound in spores ofN. crassa, we suggest that ferricrocin is stored in mycelia for inclusion into conidiospores.     

Tyr721 regulates specific binding of the CSF-1 receptor kinase insert to PI 3''-kinase SH2 domains: a model for SH2-mediated receptor-target interactions.

Efficient binding of active phosphatidylinositol (PI) 3'-kinase to the autophosphorylated macrophage colony stimulating factor receptor (CSF-1R) requires the noncatalytic kinase insert (KI) region of the receptor. To test whether this region could function independently to bind PI 3'-kinase, the isolated CSF-1R KI was expressed in Escherichia coli, and was inducibly phosphorylated on tyrosine. The tyrosine phosphorylated form of the CSF-1R KI bound PI 3'-kinase in vitro, whereas the unphosphorylated form had no binding activity. The p85 alpha subunit of PI 3'-kinase contains two Src homology (SH)2 domains, which are implicated in the interactions of signalling proteins with activated receptors. Bacterially expressed p85 alpha SH2 domains complexed in vitro with the tyrosine phosphorylated CSF-1R KI. Binding of the CSF-1R KI to PI 3'-kinase activity, and to the p85 alpha SH2 domains, required phosphorylation of Tyr721 within the KI domain, but was independent of phosphorylation at Tyr697 and Tyr706. Tyr721 was also critical for the association of activated CSF-1R with PI 3'-kinase in mammalian cells. Complex formation between the CSF-1R and PI 3'-kinase can therefore be reconstructed in vitro in a specific interaction involving the phosphorylated receptor KI and the SH2 domains of p85 alpha.     

A CD44-like endothelial cell transmembrane glycoprotein (GP116) interacts with extracellular matrix and ankyrin.

We used complementary biochemical and immunological techniques to establish that an endothelial cell transmembrane glycoprotein, GP116, is a CD44-like molecule and binds directly both to extracellular matrix components (e.g., hyaluronic acid) and to ankyrin. The specific characteristics of GP116 are as follows: (i) GP116 can be surface labeled with Na 125I and contains a wheat germ agglutinin-binding site(s), indicating that it has an extracellular domain; (ii) GP116 displays immunological cross-reactivity with a panel of CD44 antibodies, shares some peptide similarity with CD44, and has a similar 52-kDa precursor molecule, indicating that it is a CD44-like molecule; (iii) GP116 displays specific hyaluronic acid-binding properties, indicating that it is a hyaluronic acid receptor; (iv) GP116 can be phosphorylated by endogenous protein kinase C activated by 12-O-tetradecanoylphorbol-13-acetate and by exogenously added protein kinase C; and (v) GP116 and a 20-kDa tryptic polypeptide fragment of GP116 from the intracellular domain are capable of binding the membrane-cytoskeleton linker molecule, ankyrin. Furthermore, phosphorylation of GP116 by protein kinase C significantly enhances GP116 binding to ankyrin. Together, these findings strongly suggest that phosphorylation of the transmembrane glycoprotein GP116 (a CD44-like molecule) by protein kinase C is required for effective GP116-ankyrin interaction during endothelial cell adhesion events.     

Regulated degradation of ornithine decarboxylase requires interaction with the polyamine-inducible protein antizyme.

Intracellular degradation of vertebrate ornithine decarboxylase (ODC) is accelerated by polyamines, the products of the pathway controlled by ODC. Antizyme, a reversible, tightly binding protein inhibitor of ODC activity, is believed to be involved in this process. Mouse and Trypanosoma brucei ODCs are structurally similar, but the trypanosome enzyme, unlike that of the mouse, is not regulated by intracellular polyamines when expressed in hamster cells (L. Ghoda, D. Sidney, M. Macrae, and P. Coffino, Mol. Cell. Biol. 12:2178-2185, 1992). We found that mouse ODC interacts with antizyme in vitro but trypanosome ODC does not. To localize the region necessary for binding, we made a series of enzymatically active chimeric mouse-trypanosome ODCs and tested them for antizyme interaction. Replacing residues 117 to 140 within the 461-amino-acid mouse ODC sequence with the equivalent region of trypanosome ODC disrupted both antizyme binding and in vivo regulation. Formation of an antizyme-ODC complex is therefore required for regulated degradation.     

Vitamin A deficiency and inflammation: the pivotal role of secretory cells in the development of atrophic, hyperplastic and metaplastic change in the tracheal epithelium in vivo.

We showed previously that the proliferation of hamster airway secretory cells decreases during vitamin A deficiency (VAD) but later increases when submucosal inflammation develops (Virchows Arch [B] 59:231-242, 1990). This observation has important biological implications since two morphological extremes (atrophy and quiescence versus hyperplasia and hyperproliferation) are reported in the literature for VAD tracheal epithelium in vivo. In the present study, histological slides of tracheal rings from 35-day-old control and VAD hamsters (Virchows Arch [B] 45:197-219, 1984) were reviewed again. Rings from VAD hamsters were selected based on the absence or presence of a florid submucosal inflammation. Quantitative analyses were made on the cartilaginous part of rings from the anterior third of the trachea. When inflammation was absent, a mucociliary pseudostratified epithelium was, for the most part, maintained. The mitotic rate (MR, 6 h colchicine blockade) of secretory cells was markedly reduced (29-fold) but that of basal cells was not changed significantly. Moreover, cell density was not changed by VAD but ciliated cells and secretory cells were decreased and basal cells were increased, proportionally. We call this "minimal morphological change." Thinning (atrophy) of the minimally changed epithelium was associated with focal cell sloughing. Small scattered foci of epidermoid metaplasia (multiple layers of highly keratinized cells which were extremely flat, so that the epithelium was thin and attenuated) were also seen. We call this "atrophic epidermoid metaplasia." When inflammation was present, hyperplastic changes (stratification and epidermoid metaplasia) predominated and cells were in mitosis at all epithelial levels (low, middle, superficial) except in the most superficial (terminally differentiated) squames. The tracheal epithelium was thickened and hypercellular. The cells were piled up at the stratified lesions, and epithelial height, cell density and epithelial MR were significantly increased compared with the non-inflamed VAD epithelium. The effects of VAD and inflammation on cell proliferation were analyzed further by studying 7 h bromodeoxyuridine (BrdU) labelling patterns of cells in VAD tracheal epithelium, with and without submucosal inflammation. In addition, inflammation was induced in "minimally changed epithelium" by mild mechanical injury. The BrdU labelling patterns confirmed that DNA synthesis by secretory cells is reduced markedly by VAD. However, this suppression is overidden by the influx of inflammatory cells (the nature of the stimulus is unknown). The results indicate that the morphological contrasts (atrophy and hyperplasia) seen in the trachea during VAD in vivo are related to extremes in proliferation rates of tracheal secretory cells, regulated by VAD alone (minimal replication) and by inflammation (maximal replication).     

头端延髓腹外侧区注射5—羟色胺对应激性高血粘度...

Experiments were carried out on 62 wistar rats. The hyperviscosity and elevation of blood pressure were induced by hanging and restraining the rats with their four limbs tied on a frame. It was found that microinjection of 5-HT (25 micrograms/10 microliters) into the 4th ventricle of the brain or bilateral microinjection of 5-HT (4 micrograms/0.5 microliters/site) into rostral ventrolateral medulla (rVLM) reduced stress-induced hyperviscosity (p < 0.01) and elevation of blood pressure (p < 0.01). The effect of 5-HT injected into the 4th ventricle or rVLM was blocked by bilateral microinjection of cinanserine (4 micrograms/0.5 microliter/site) into rVLM. These results suggest that microinjection of 5-HT into 4th ventricle and rVLM could reduce stress-induced hyperviscosity and elevation of blood pressure and these effects were probably mediated via 5-HT receptors in the rVLM.