The Ca2(+)-dependent actin filament-severing activity of 74-kDa protein (adseverin) resides in its NH2-terminal half.

Calcium sensitive actin severing protein, adseverin, with Mr 74,000, was cleaved into two fragments of Mr 42,000 and Mr 39,000 by V8 protease and trypsin, and both fragments were purified by high performance (pressure) liquid chromatography ion-exchange column chromatography. To understand how adseverin can sever actin filaments, we identified the actin-binding domains. The NH2 termini of native adseverin and the Mr 42,000 fragment were confirmed to be blocked by amino acid sequencing. Twelve amino acids of the Mr 39,000 fragment were sequenced from the NH2 terminus; the sequence of this part had a homology to the hinge region between segments 3 and 4 of gelsolin and villin. Thus, the Mr 42,000 fragment is the NH2-terminal half (N42), and the Mr 39,000 fragment is the COOH-terminal half (C39). Each fragment was examined for actin-severing, -nucleating, -capping, and phospholipid binding activities with and without calcium. N42 contained a calcium-dependent actin-severing activity regulated by phospholipid. C39 bound to G-actin in a calcium-dependent manner, but had no severing activity. The sequence homology and similar functional domain structure suggest a common structural basis for the calcium- and phospholipid-regulated actin-severing properties shared by adseverin, gelsolin, and villin.     

Control of K+ channels by G proteins

Heterotrimeic G proteins are thought to couple receptors to ionic channels via cytoplasmic mediators such as cGMP in the case of retinal rods, cAMP in the case of olfactory cells, and the cAMP cascade in the case of cardiac myocytes. G protein-mediated second messenger effects on K⁺ channels are dealt with elsewhere in this series. Recently, membrane-delimited pathways have been uncovered and an hypothesis proposed in which the subunits of G proteins directly couple receptors to ionic channels, particularly K⁺ channels. While direct coupling has not been proven, the membrane-delimited nature has been established for specific G proteins and their specific K⁺ channel effectors.     

A molecular genetic linkage map of mouse chromosome 10, including theMyb,S100b,Pah, Sl,andIfg genes

Restriction endonuclease fragment length variations (RFLV) on mouse chromosome 10 were detected in four genes, namely, the Myb protooncogene (Myb) and the genes for S100 beta protein (S100b), phenylalanine hydroxylase (Pah), and interferon-gamma (Ifg). RFLV were found in restriction patterns generated with BamHI for Myb, in those generated with BglII for S100b, in those generated with EcoRV for Pah, and in those generated with TaqI for Ifg. A multipoint backcross was carried out by the mating (129/Sv-Sl/+ x MOL-MIT)F1 x 129/SvJ(-)+/+. The Sl mutation has phenotypic effects which include deficiencies in pigment cells, germ cells, and blood cells. The following order of genes was derived from the results of the multipoint backcross, with distances between genes in parentheses: centromere--Myb--(34.9 cM)--S100b--(8.5 cM)--Pah--(8.5 cM)--Sl--(12.3 cM)--Ifg--telomere. Most laboratory strains and two strains of Mus musculus domesticus of wild origin carry the Myba, S100a, Paha, and Ifga alleles. In contrast, a strain of M. m. musculus, two strains of M. m. yamashinai, and two strains of M. m. molossinus carry the Mybb, S100b, Pahb, and Ifgb alleles. Other strains of wild origin carry various combinations of these alleles.     

Growth-stimulatory effects of androgen, high concentration of glucocorticoid or fibroblast growth factors on a cloned cell line from Shionogi carcinoma 115 cells in a serum-free medium

The effects of various kinds of growth factors or steroids on the proliferation of Shionogi carcinoma 115 (SC115) cells were investigated in cell culture. In a serum-free medium [Ham's F-12:Eagle's minimum essential medium (1:1, vol/vol) containing 0.1% bovine serum albumin], the proliferation of SC-3 cells (a cloned cell line from SC115 cells) estimated by [3H]thymidine incorporation into DNA and cell number reached a plateau at 10(-8) M testosterone (up to 200-fold), 10(-7) M dexamethasone (up to 30-fold) or 1 ng/ml of fibroblast growth factors (FGF; up to 50-fold). However, the proliferation in the serum-free medium was not significantly stimulated by the addition of low to very high concentrations of progesterone, oestradiol-17 beta, epidermal growth factor, platelet derived growth factor or insulin; transforming growth factor beta slightly stimulated the growth (up to 5-fold) but markedly inhibited the growth stimulation induced by testosterone. Furthermore, an epithelial appearance of SC-3 cells grown in the absence of growth factors or steroids was changed to a fibroblast-like appearance only by the addition of testosterone, high concentrations of dexamethasone or FGF. By investigating various kinds of growth factors or steroids, the present study demonstrates that androgen, high concentration of glucocorticoid or FGF alone significantly stimulates the proliferation of SC-3 cells with a change of morphology in the serum-free medium.     

Inhibition of Maitotoxin-Induced Ca2+ Influx in Rat Glioma C6 Cells by Brevetoxins and Synthetic Fragments of Maitotoxin

Abstract: ⁴⁵Ca²⁺ influx in rat glioma C6 cells induced by 0.3 n M maitotoxin (MTX) was markedly inhibited by brevetoxin A (PbTx1) and brevetoxin B (PbTx2), with EC₅₀ values of 16 and 13 µ M , respectively. This inhibition was observed immediately after addition of MTX when monitored with fura-2, which suggests that PbTx2 directly blocks the action of MTX. This blockade by PbTx2 was not affected by tetrodotoxin, which excludes the involvement of voltage-sensitive sodium channels. The depolarizing effects of these brevetoxins were also not a likely cause of this inhibition, because melittin, a channel-forming peptide, did not significantly block MTX-induced ⁴⁵Ca²⁺ influx. Instead, this inhibition was thought to be mediated by blockade of an MTX-binding site by the brevetoxins, based on the fact that these toxins, particularly PbTx2, closely mimic the partial structure of MTX. Synthetic fragments of MTX corresponding to the hydrophilic EF-GH rings (200 µ M ) and LM-NO rings (500 µ M ) of MTX significantly reduced MTX-elicited Ca²⁺ influx. The observation that the effects of MTX were inhibited by structural mimics of both its hydrophobic and hydrophilic portions implies that both portions of the MTX molecule recognize its target.     

Continuous Treatment with Morphine Increases Diazepam Binding Inhibitor mRNA in Mouse Brain

Abstract: Effects of acute and chronic morphine treatment on the expression of diazepam binding inhibitor (DBI) mRNA in the mouse brain were examined. Cerebral DBI mRNA expression significantly increased in morphine-dependent mice, and this increase is more remarkable in morphine-withdrawn mice, whereas a single administration of morphine (50 mg/kg) produced no changes in the expression. Simultaneous administration of naloxone (3 mg/kg) with morphine completely abolished the increase in cerebral DBI mRNA expression observed in morphine-dependent and -withdrawn mice. These results indicate that a chronic functional interaction between morphine and opioid receptors has a critical role in increases in DBI mRNA expression.     

The AATPAP sequence is a very efficient signal for O-glycosylation in CHO cells

The peptide signal sequence for protein O-glycosylation is not fully characterized, although a recent in vitro study proposed that the sequence motif, XTPXP, serves as a signal for mucin-type O-glycosylation. Here, we show that the AATPAP sequence acts as an efficient O-glycosylation signal, in vivo. A secreted fibroblast growth factor (secFGF) was used as a model to analyze glycosylation and its effects on the biological activity of FGF. Two constructs encoding [AATPAP]secFGF in which AATPAP was introduced at the N- or C-terminus of secFGF were constructed in a eukaryotic expression vector. [AATPAP]secFGF proteins were then expressed in Chinese hamster ovary (CHO) cells and secreted into the surrounding medium, primarily as modified forms sensitive to sialidase but not to peptide N-glycosidase F. The modifying groups were not seen when the AATPAP sequence was converted to AAAPAP or when [AATPAP]secFGF was expressed in mutant cells incapable of UDP-GalNAc biosynthesis. The results indicate that the modifying groups were mucin-type O-glycans and that the AATPAP served as an efficient O-glycosylation signal sequence. The O-glycosylated forms of [AATPAP]secFGF were as mitogenic toward human vascular endothelial cells as unmodified secFGF, suggesting that introduction of the signal into biologically active polypeptides is a promising approach with which O-glycosylation may be achieved without affecting original activity.     

Dynamic interaction between the tongue and soft palate during obstructive apnea in anesthetized patients with sleep-disordered breathing.

Little is known about the mechanisms of persistence of obstructive apnea. Structurally, the dorsum of the tongue locates anterior to the soft palate. On the basis of the observation of posterior displacement of the tongue during obstructive apnea, we hypothesized that the dorsum of the tongue pushes the anterior wall of the soft palate posteriorly during inspiratory efforts, maintaining closure at the retropalatal airway. To test this hypothesis, we measured the pressure between dorsum of the tongue and anterior wall of the soft palate (PT&P) during experimentally induced obstructive apneas in anesthetized patients with sleep-disordered breathing. P(T&P) changes during the obstruction significantly depended on collapsibility of the retroglossal airway. Progressive increase in the P(T&P) during obstructive apnea was observed only in patients with highly collapsible retroglossal airways. Significant increase in the P(T&P) during inspiratory effort in accordance with positive deflection pattern of P(T&P) tracing was evident in the patients with highly collapsible retroglossal airways. The results indicate significant dynamic interaction between the tongue and soft palate during both obstructive apnea and each inspiratory effort, possibly maintaining closure at the retropalatal airway.