Mechanism of membrane damage induced by the amphipathic peptides gramicidin S and melittin

The action of gramicidin S and melittin on human erythrocytes, Staphylococcus aureus and Escherichia coli was studied as an extension of the previous study (Katsu, T., Ninomiya, C., Kuroko, M., Kobayashi, H., Hirota, T. and Fujita, Y. (1988) Biochim. Biophys. Acta 939, 57-63). These amphipathic peptides stimulated the release of membrane phospholipids outside cells in a concentration range causing permeability change. The shape change of erythrocytes from normal discoid to spiculate form was observed just prior to the release of membrane components. We have proposed the following action mechanism of gramicidin S and melittin. The peptide molecules were predominantly accumulated in the outer half of the bilayer, deforming the erythrocyte cell into crenature. A large accumulation made the membrane structure unstable, resulting in the release of membrane fragments and the simultaneous enhancement of permeability. The action mechanism of these peptides was compared with that of simple surfactants.     

Novel migrating mouse neural crest cell assay system utilizing P0-Cre/EGFP fluorescent time-lapse imaging

Background

Heparan sulfate (HS) is present on the surface of virtually all mammalian cells and is a major component of the extracellular matrix (ECM), where it plays a pivotal role in cell-cell and cell-matrix cross-talk through its large interactome. Disruption of HS biosynthesis in mice results in neonatal death as a consequence of malformed lungs, indicating that HS is crucial for airway morphogenesis. Neonatal mortality (~50%) in newborns with congenital diaphragmatic hernia (CDH) is principally associated with lung hypoplasia and pulmonary hypertension. Given the importance of HS for lung morphogenesis, we investigated developmental changes in HS structure in normal and hypoplastic lungs using the nitrofen rat model of CDH and semi-synthetic bacteriophage ('phage) display antibodies, which identify distinct HS structures.

Results

The pulmonary pattern of elaborated HS structures is developmentally regulated. For example, the HS4E4V epitope is highly expressed in sub-epithelial mesenchyme of E15.5 - E17.5 lungs and at a lower level in more distal mesenchyme. However, by E19.5, this epitope is expressed similarly throughout the lung mesenchyme. We also reveal abnormalities in HS fine structure and spatiotemporal distribution of HS epitopes in hypoplastic CDH lungs. These changes involve structures recognised by key growth factors, FGF2 and FGF9. For example, the EV3C3V epitope, which was abnormally distributed in the mesenchyme of hypoplastic lungs, is recognised by FGF2.

Conclusions

The observed spatiotemporal changes in HS structure during normal lung development will likely reflect altered activities of many HS-binding proteins regulating lung morphogenesis. Abnormalities in HS structure and distribution in hypoplastic lungs can be expected to perturb HS:protein interactions, ECM microenvironments and crucial epithelial-mesenchyme communication, which may contribute to lung dysmorphogenesis. Indeed, a number of epitopes correlate with structures recognised by FGFs, suggesting a functional consequence of the observed changes in HS in these lungs. These results identify a novel, significant molecular defect in hypoplastic lungs and reveals HS as a potential contributor to hypoplastic lung development in CDH. Finally, these results afford the prospect that HS-mimetic therapeutics could repair defective signalling in hypoplastic lungs, improve lung growth, and reduce CDH mortality.     

Phosphorylation of smg p21, a ras p21-like GTP-binding protein, by cyclic AMP-dependent protein kinase in a cell-free system and in response to prostaglandin E1 in intact human platelets

We have separated multiple small Mr GTP-binding proteins (G proteins) from bovine brain membranes by several column chromatographies and purified to near homogeneity four of them, including a novel Mr 24,000 G protein (smg p25A), a novel Mr 22,000 G protein (smg p21), the rho protein (rho p20), and the c-Ki-ras protein (c-Ki-ras p21). Among these small Mr G proteins, only smg p21 is phosphorylated stoichiometrically by cAMP-dependent protein kinase (protein kinase A), and c-Ki-ras p21 is phosphorylated to a small extent by protein kinase A in a cell-free system. None of smg p25A, rho p20, and other partially purified small Mr G proteins is phosphorylated by protein kinase A. Neither smg p21 nor other small Mr G proteins are phosphorylated by protein kinase C. About 1 mol of phosphate is maximally incorporated into 1 mol of smg p21 by protein kinase A. Only serine residue(s) are phosphorylated. The guanosine 5'-3-O-(thio) triphosphate (GTP gamma S)-bound and GDP-bound forms of smg p21 are phosphorylated with the same reaction velocity. The phosphorylation of smg p21 affects neither its GTP gamma S-binding nor GTPase activity. smg p21 is found in human platelets, and this human platelet smg p21 is also phosphorylated by protein kinase A at the same site(s) as bovine brain smg p21 in a cell-free system. When intact human platelets are stimulated by prostaglandin E1 known to elevate the cAMP level, four proteins with apparent Mr values of 240,000, 50,000, 24,000, and 22,000 are phosphorylated. These four proteins are also phosphorylated by the action of dibutyryl cAMP but not by the action of thrombin, Ca2+ ionophore A23187, or 12-O-tetradecanoylphorbol-13-acetate. Among the four proteins, the Mr 22,000 protein is identified as smg p21. The site(s) of phosphorylation of smg p21 by protein kinase A in a cell-free system are identical to that phosphorylated in response to prostaglandin E1 in intact platelets. These results indicate that among many small Mr G proteins, smg p21 is selectively phosphorylated by protein kinase A and that this G protein is also phosphorylated by this protein kinase in response to prostaglandin E1 in intact human platelets.     

Expression of messenger RNA for gonadotropin receptor in the granulosa layer during the ovulatory cycle of hens.

The present experiments were conducted to evaluate the mRNA levels of luteinizing hormone receptor (LHR) and follicle-stimulating hormone receptor (FSHR) in granulosa layers during the ovulatory cycle of hens, in relation to the release of LH and steroid hormones. After the release of LH, progesterone (P4) and estradiol-17beta (E2), found 4-5 h before ovulation, LHR and FSHR mRNA levels were observed to decrease in the granulosa layers of the largest (F1) and second largest (F2) preovulatory follicles, with the greatest in the LHR mRNA level of F1. P4 concentrations in the granulosa layers of F1 and F2 increased 4-5 h before ovulation, with greater in F1 than in F2. F2 concentrations in the theca layers were greater in F2 than in F1 throughout the ovulatory cycle. Also, the injection of ovine LH caused decreases in the mRNA levels of LHR and FSHR in the granulosa layers. However, these decreases were abolished by the injection of aminoglutethimide, an inhibitor of steroid synthesis. These results suggest that in hen granulosa cells, the mRNA levels of not only LHR but also FSHR are down-regulated by LH and the down-regulation may be mediated steroid hormones.     

Bombesin evoked acetylcholine release from the guinea pig antrum

Bombesin induced contraction and acetylcholine (ACh) release of the longitudinal muscle strip of the guinea pig antrum were examined using the standard organ bath technique and the superfusion system. Bombesin increased frequency and tonus of rhythmic contraction in a dose dependent manner (10(-10)M - 10(-7)M). The effects of bombesin on frequency of contraction were not affected by atropine, propranolol, phentolamine, hexamethonium and tetrodotoxin. The effects on tonus, on the other hand, were significantly reduced by atropine, and the dose response curve to bombesin was shifted to the right. There was a remarkable increase of 3H-ACh release by the superfusion of bombesin (10(-8)M), which was almost completely abolished in Ca-free medium, but not affected by hexamethonium and tetrodotoxin. These results suggest that mechanism of bombesin effects on frequency is different from that on tonus; frequency response to bombesin is not dependent on autonomic nervous system but due to a direct effect on smooth muscle cells, whereas tonic response to the peptide is partly mediated by ACh release via a mechanism independent of sodium spike.     

Forced expression of FLO11 confers pellicle-forming ability and furfural tolerance on Saccharomyces cerevisiae in ethanol production

We constructed a plasmid that expresses FLO11 encoding a cell surface glycoprotein of Saccharomyces cerevisiae under the control of a constitutive promoter. This plasmid conferred pellicle-forming ability on the non-pellicle-forming industrial strain of S. cerevisiae at the air–liquid interface of the glucose-containing liquid medium. The induced pellicle-forming cells exhibited tolerance to furfural, which is a key toxin in lignocellulosic hydrolysates, in ethanol production.     

Separate cis-acting DNA elements control cell type- and tissue-specific expression of collagen binding molecular chaperone HSP47

HSP47 is a collagen-binding heat shock protein and is assumed to act as a molecular chaperone in the biosynthesis and secretion of procollagen. As the synthesis of HSP47 is closely correlated with that of collagen in various cell lines and tissues, we performed a promoter/reporter assay using HSP47-producing and nonproducing cells. 280 base pairs (bp(s)) of upstream promoter were shown to be necessary for the basal expression but not to be enough for the cell type-specific expression. When the first and the second introns were introduced downstream of this 280-bp region, marked up-regulation of the reporter activity was observed in HSP47-producing cells but not in nonproducing cells. This was confirmed in transgenic mice by staining the lacZ gene product under the control of the 280-bp upstream promoter and the introns. Staining was observed in skin, chondrocytes, precursor of bone, and other HSP47/collagen-producing tissues. A putative Sp1-binding site at -210 bp in the promoter, to which Sp3 and an unidentified protein bind, was shown to be responsible for this up-regulation when combined with the introns. However no difference in the binding to this probe was observed between HSP47-producing and nonproducing cells. The responsible region for cell type-specific up-regulation was found to be located in a 500-bp segment in the first intron. On electrophoresis mobility shift assay using this 500-bp probe, specific DNA-protein complexes were only observed in HSP47-producing cell extracts. These results suggest that two separate elements are necessary for the cell type-specific expression of the hsp47 gene; one is a putative Sp1-binding site at -210 bp necessary for basal expression, and the other is a 500-bp region within the first intron, required for cell type-specific expression.