Multigranular exocytosis induced by phospholipase A2-activators,melittin and mastoparan,in rat anterior pituitary cells

Summary Effects of phospholipase A₂-activators, melittin and mastoparan, on rat anterior pituitary cells were studied by use of the electron microscope. Rat anterior pituitaries were incubated in HEPES buffer containing 20 g/ml of melittin or the same dose of mastoparan for 5 min, 10 min and 20 min. Features indicating discharge of granule contents by exocytosis were increased with time, and the simultaneous extrusion of a number of secretory granules, named multigranular exocytosis, was often recognized in addition to single-granule exocytosis at 10 min and 20 min. Most membrane pits, where the multigranular exocytosis as well as the single-granule exocytosis occurred, were coated. Moreover, a large number of vesicles coated or noncoated were distributed near the trans side of the Golgi apparatus of melittin-treated or mastoparan-treated cells after 20 min. These vesicles might be related to membrane internalized from the excess surface membrane derived from the limiting membrane of exocytosed granules. These observations indicate that phospholipase A₂-activators induce hormone release involving membrane fusion between limiting membranes of secretory granules, and between granulelimiting membrane and plasma membrane in rat anterior pituitary cells.This study was supported by grants from the Japan Ministry of Education     

Electrofusion of giant protoplasts of Pleurotus cornucopiae

Summary Giant protoplasts of Pleurotus cornucopiae were fused, using the glass microelectrode electrofusion technque; the percentage fusion achieved was 70%. To induce fusion, Ca²⁺ was necessary, a 10 mM concentration giving the best result. Polyethylene glycol 4000 (PEG) promoted fusion but also increased the adhesion of protoplasts, which caused them to be irreversibly attached to the electrodes. Fusion was always completed within 1 min after a single electrical pulse had been applied. The fused protoplast was isolated with a glass micropipette and was found to regenerate into a colony.     

Extracellular Production of Yeast Protease

The groEL gene of the alkaliphilic Bacillus sp. strain C-125 was cloned in Escherichia coli and sequenced. The groEL gene encoded a polypeptide of 544 amino acids and was preceded by the incomplete groES gene, lacking its 5′-end. The sequence of the derived amino acids was 87.5% identical to that of B. subtilis, 85.4% identical to that of B. stearothemophilus, and 60.9% identical to that of E. coli. The GroEL protein was expressed in E. coli. Purified GroEL protected yeast a-glucosidase from irreversible aggregation at a high temperature and the addition of Mg-ATP was essential for reactivation of the a-glucosidase. The addition of E. coli GroES increased recovery of the enzyme activity, indicating that C-125 GroEL could function in coordination with E. coli GroES.     

Affinity Chromatography of Acid Proteases on SPI Coupled to PAB-Cellulose

Tastes of Leu-Lys-Tyr (LKY) analogues, a series of potent angiotensin I converting enzyme (ACE) inhibitory peptides were evaluated. Some of these analogues were found to be sweet, such as Val–Lys-Tyr and Ala-Orn-Tyr. Furthermore, the structural requirements for sweetness or decreasing the bitterness were investigated by considerations of the structure–taste relationship with LKY analogues.     

N-acetyl-d-glucosamine induces the expression of multidrug exporter genes, mdtEF, via catabolite activation in Escherichia coli

The expression of MdtEF, a multidrug exporter in Escherichia coli, is positively controlled through multiple signaling pathways, but little is known about signals that induce MdtEF expression. In this study, we investigated compounds that induce the expression of the mdtEF genes and found that out of 20 drug exporter genes in E. coli, the expression of mdtEF is greatly induced by N-acetyl-d-glucosamine (GlcNAc). The induction of mdtEF by GlcNAc is not mediated by the evgSA, ydeO, gadX, and rpoS signaling pathways that have been known to regulate mdtEF expression. On the other hand, deletion of the nagE gene, encoding the phosphotransferase (PTS) system for GlcNAc, prevented induction by GlcNAc. The induction of mdtEF by GlcNAc was also greatly inhibited by the addition of cyclic AMP (cAMP) and completely abolished upon deletion of the cAMP receptor protein gene (crp). Other PTS sugars, glucose and d-glucosamine, also induced mdtEF gene expression. These results suggest that mdtEF expression is stimulated through catabolite control.     

Nitric oxide-producing microglia mediate thrombin-induced degeneration of dopaminergic neurons in rat midbrain slice culture

Activated microglia are considered to play important roles in degenerative processes of midbrain dopaminergic neurons. Here we examined mechanisms of neurotoxicity of thrombin, a protease known to trigger microglial activation, in organotypic midbrain slice cultures. Thrombin induced a progressive decline in the number of dopaminergic neurons, an increase in nitric oxide (NO) production, and whole tissue injury indicated by lactate dehydrogenase release and propidium iodide uptake. Microglia expressed inducible NO synthase (iNOS) in response to thrombin, and inhibition of iNOS rescued dopaminergic neurons without affecting whole tissue injury. Inhibitors of mitogen-activated protein kinases (MAPKs) such as extracellular signal-regulated kinase (ERK), p38 MAPK and c-Jun N-terminal kinase (JNK) attenuated thrombin-induced iNOS induction and dopaminergic cell death. Whole tissue injury was also attenuated by inhibition of ERK and p38 MAPK. Moreover, depletion of resident microglia from midbrain slices abrogated thrombin-induced NO production and dopaminergic cell death, but did not inhibit tissue injury. Finally, antioxidative drugs prevented thrombin-induced dopaminergic cell death without affecting whole tissue injury. Hence, NO production resulting from MAPK-dependent microglial iNOS induction is a crucial event in thrombin-induced dopaminergic neurodegeneration, whereas damage of other midbrain cells is MAPK-dependent but is NO-independent.     

Patterns of tail breakage in the ladder snake (Rhinechis scalaris) reflect differential predation pressure according to body size

Predator-prey interactions are key factors in the evolution of defensive tactics. In snakes, shy organisms from which direct evidence of predator-prey interactions is difficult to obtain, injuries are potential indicators of both the nature and frequency of interactions. We studied the incidence of tail breakage and body scarring in the ladder snake, Rhinechis scalaris, an actively foraging Mediterranean snake, and tested several hypotheses that link body injuries and snake life-history traits, mainly under sexual and ontogenetic aspects. Evidence is presented supporting an ontogenetic shift in the frequency of tail breakage, with the incidence of tail loss increasing as a logistic function of snake size. We relate this finding to the adaptive significance of ontogenetic shifts in dorsal pattern and the reaction of snakes to approaching predators; small individuals are more likely to remain immobile than are medium to large individuals, the former aided by a concealing dorsal pattern with transverse lines, and the latter by a striped pattern. This species exhibited sex differences in body scarring but not in tail breakage, nor did we encounter evidence to suggest that snakes experience multiple tail breaks over time, thus failing to support the sexual difference and multiple tail breakage hypotheses. Moreover, we failed to find a lower frequency of body scarring than tail breakage; hence, our results do not afford evidence that frequency of tail breakage represents an inefficiency of predators in catching or dispatching ophidian prey.     

Oriented structure of pectic polysaccharides in pea epidermal cell walls

Polarized infrared absorption spectra of film specimens of theepidermal cell wall of the third internode of pea stems wererecorded before and after treatment with endopolygalacturonase(endo-PG) and endo-pectin lyase (endo-PL). The spectra showedthat the pectic polysaccharides solubilized with endo-PG wereessentially the same as those solubilized with endo-PL. Thedegree of esterification of the pectic polysaccharides was about20%, and their major sugar components were uronic acids (32.8%),arabinose (48.1%) and galactose (19.2%). The polarized infraredspectra showed that pectic polysaccharides have an orientedstructure in cell walls with their molecular chains orientedpreferentially parallel to the direction of cell elongation. ¹Present address: Research and Development, Kanzaki Paper Mfg.Co., Ltd., Amagasaki, Hyogo 660, Japan. ²Present address: Wakayama Research Laboratories, Kao Soap Co.,Ltd., Wakayama 640-91, Japan. (Received June 28, 1980; )