Effects of ACTH, dexamethasone, adrenalectomy and stress on cAMP content of adenohypophysis and adrenals in rats.

The effects of adrenalectomy, ether-laparotomy stress and in vivo administration of either ACTH or dexamethasone on the cAMP levels in the anterior pituitary and the adrenal glands were investigated in male rats. After adrenalectomy or ether-laparotomy stress, an increase of the pituitary cAMP levels was observed. A prior administration of dexamethasone failed to inhibit the increase of the pituitary cAMP levels. Acute administration of dexamethasone increased the cAMP levels in the pituitary. Chronic administration of dexamethasone decreased the cAMP levels in the pituitary, but ACTH did not. These data suggest that cAMP might be involved in the mediation of the hypothalamo-pituitary-adrenal axis.     

Normal skin cells in vitro

Major cells in skin are epidermal cells in epidermis and fibroblasts in dermis. These cells can be isolated as a relatively pure population from the tissues using proteases and chelating agents. In this review we describe the way of culture where these two kinds of cells express normal function as they do in vivo. 1) It is important to consider the polarity of epidermal cell membranes in the transport of nutrients and metabolites when the cells are to be cultured in a healthy state in a long period. Epidermals cells expressed their normal polarities when cultured on a porous thin film of collagen and bathed on both sides (apical and basal) in culture media. 2) It is important to consider the interactions of fibroblasts with collagen when normal morphology and physiology are expected to be expressed in the cell in culture. Collagen affected the morphology of the cell and profoundly decreased the rate of DNA synthesis. We present a hypothesis which explains the fibronectin-independent interaction of fibroblasts with collagen. 3) It is important to consider the interactions between fibroblasts and epidermal cells when normal physiology of the skin as a whole is expected to be expressed in vitro, because exchange of information between them control their metabolic activities and functions. In this review, two examples for this exchange are presented: cell growth and collagenolysis.     

A human enzyme that liberates uracil from DNA

An enzyme activity which acts specifically on uracil-containing DNA was found in human placenta and cultured fibroblasts. The enzyme liberates uracil from DNA in the presence of EDTA at pH 7.5. Almost equal levels of the activity were found in normal and xeroderma pigmentosum cell lines (complementation group A).     

Separation and characterization of diastereoisomeric oligonucleotide.

For developing the antisense method, two types of oligonucleotides suitable for antisense molecules were explored for their essential properties. One is oligonucleoside phosphorothioate, and all their possible R/S diastereoisomers were separated and purified by reversed phase liquid chromatography. Isolated diastereoisomers have been investigated for the correlation between their configuration and hybridization manner. Separation, R/S characterization and spectroscopical properties of these oligonucleotides will be discussed.     

Isolation and Biological Characterization of a Measles Virus-Like Agent from the Brain of an Autopsied Case of Subacute Sclerosing Panencephalitis (SSPE)

Isolation of a cytopathic agent causing formation of syncytial giant cells in co-cultivated Vero cells from the brain of an autopsied case of subacute sclerosing panencephalitis (SSPE) is reported. The syncytia usually autolyzed from the center after growing to 1 to 2 mm in diameter and then detached from the culture vessels, and finally made macroscopically recognizable round plaques on the monolayer under liquid overlay. The agent was identified serologically as an agent related to measles virus, by both immunofluorescent tests and plaque reduction tests using anti-measles sera. However, the infected cells did not produce either virions or hemagglutinin, and failed to show hemadsorption and hemolysis of African green monkey red cells even after the 55th passage through Vero cells. Newborn mice, adult mice and hamsters showed neurologic signs after intracerebral inoculations of the infected cells, and most of them died from acute encephalitis. Guinea pigs were unsusceptible. From the brain of the animals with neurologic signs, a similar agent to the inoculated one was recovered. The new isolate appears to be a strain closely related to measles virus on the basis of serology, and was designated as SSPE-“Kitaken-1” strain.     

The presence of l-2,4-diaminobutyric acid decarboxylase activity in Vibrio species: A new biosynthetic pathway for 1,3-diaminopropane

Abstract A new enzyme activity, which catalyzes decarboxylation of l -2,4-diaminobutyric acid (DABA) to yield 1,3-diaminopropane (DAP), has been found in dialyzed crude extracts prepared from Vibrio alginolyticus . The pH optimum for the activity was 8.0–8.5, and the enzyme showed a pyridoxal 5'-phosphate (PLP) requirement. Mg²⁺ caused about 30% stimulation in activity. The enzyme was active to only l -DABA among the diamino acids examined, and the K _m value for l -DABA was 0.13 mM. Ammonium sulfate fractionation of a dialyzed crude extract followed by HPLC separation allowed us to conclude that this enzyme differed from the decarboxylase which occurs in Vibrio spp. to produce norspermidine (Nspd) for carboxynorspermidine (C-Nspd) having a moiety similar in structure to DABA. The same enzyme activity was detected in several other Vibrio species.     

A Secreted Protein with Plant-Specific Cysteine-Rich Motif Functions as a Mannose-Binding Lectin That Exhibits Antifungal Activity

Plants have a variety of mechanisms for defending against plant pathogens and tolerating environmental stresses such as drought and high salinity. Ginkbilobin2 (Gnk2) is a seed storage protein in gymnosperm that possesses antifungal activity and a plant-specific cysteine-rich motif (domain of unknown function26 [DUF26]). The Gnk2-homologous sequence is also observed in an extracellular region of cysteine-rich repeat receptor-like kinases that function in response to biotic and abiotic stresses. Here, we report the lectin-like molecular function of Gnk2 and the structural basis of its monosaccharide recognition. Nuclear magnetic resonance experiments showed that mannan was the only yeast (Saccharomyces cerevisiae) cell wall polysaccharide that interacted with Gnk2. Gnk2 also interacted with mannose, a building block of mannan, with a specificity that was similar to those of mannose-binding legume lectins, by strictly recognizing the configuration of the hydroxy group at the C4 position of the monosaccharide. The crystal structure of Gnk2 in complex with mannose revealed that three residues (asparagine-11, arginine-93, and glutamate-104) recognized mannose by hydrogen bonds, which defined the carbohydrate-binding specificity. These interactions were directly related to the ability of Gnk2 to inhibit the growth of fungi, including the plant pathogenic Fusarium spp., which were disrupted by mutation of arginine-93 or the presence of yeast mannan in the assay system. In addition, Gnk2 did not inhibit the growth of a yeast mutant strain lacking the α1,2-linked mannose moiety. These results provide insights into the molecular basis of the DUF26 protein family.Plants have evolved to survive in environments that expose them to various stress factors. Being sessile, plants have developed specific mechanisms for defending against plant pathogens and responding to environmental stress conditions. These mechanisms serve to minimize damage while conserving valuable resources for growth and reproduction. For the biotic stresses caused by pathogen infections, plants have a variety of defense mechanisms such as hypersensitive responses, reinforcement of cell walls, and synthesis of phytoalexins and antifungal proteins (Hammond-Kosack and Jones, 1996; de Wit, 2007; Hamann, 2012). Abiotic stresses such as drought and high salinity also have adverse effects on plant physiology and metabolism. Plant cells respond and adapt to these adverse conditions by sensing them through receptor proteins on the plasma membrane and by mechanisms such as abscisic acid (ABA) signaling (Umezawa et al., 2010; Miyakawa et al., 2013; Osakabe et al., 2013). In fact, there is intricate cross talk among these stress responses on multiple molecular levels (Atkinson and Urwin, 2012).The genes coding the plant-specific Cys-rich motif (domain of unknown function26 [DUF26]) make up a large gene family, and the motif was initially found in the extracellular region of cysteine-rich receptor-like kinases (CRKs) and Cys-rich secretory proteins from Arabidopsis (Arabidopsis thaliana; Chen, 2001). Several CRKs are involved in resistance to bacterial and fungal pathogens, hypersensitive response-related cell death, oxidative stress responses, and salicylic acid-dependent defense pathways (Czernic et al., 1999; Du and Chen., 2000; Ohtake et al., 2000; Chen et al., 2003; Acharya et al., 2007; Wrzaczek et al., 2010; Rayapuram et al., 2012). A recent study also showed that some CRKs regulate ABA signaling and osmotic stress responses (Tanaka et al., 2012). In addition, another DUF26-containing secretory protein, Oryza sativa root meander curling, was implicated in the salt stress response in rice (Oryza sativa; Zhang et al., 2009). These findings support the idea that the DUF26 proteins have evolved to cope with various biotic and abiotic stresses in plants. However, the molecular basis of DUF26 proteins remains unclear in spite of their physiological significance.In a previous study, we identified a secreted DUF26 protein with antifungal activity, ginkbilobin2 (Gnk2), from gymnosperm Ginkgo biloba seeds (Sawano et al., 2007). Gnk2 consists of 108 amino acids as a mature protein and inhibits the growth of phytopathogenic fungi such as Fusarium oxysporum. This antifungal protein shows no sequence similarity to other antifungal proteins (Sawano et al., 2007) and thus exhibits a structure quite distinct from theirs (Miyakawa et al., 2009). Gnk2 orthologs are also found as embryo abundant proteins in two other gymnosperm species: Picea abies and Picea glauca (Sawano et al., 2007). Their sequences are not homologous to any classes of late-embryogenesis abundant proteins (Dong and Dunstan, 1999; Hong-Bo et al., 2005). In addition, the gene expressions of embryo abundant proteins are not affected by ABA, a stimulator for somatic embryo maturation, unlike the gene expressions of late-embryogenesis abundant proteins (Dong and Dunstan, 1999). These findings raise the possibility that Gnk2-like proteins function in biotic stress responses and in seed development via a specific molecular mechanism.Many pathogenesis-related proteins with antifungal activity initially target the cell wall components of fungi, including the cell wall structure and plasma membrane (Selitrennikoff, 2001). Using a NMR technique, we show that mannan is the only cell wall component with which Gnk2 interacts. Our series of structural and biochemical experiments revealed that Gnk2 functions as a lectin and that its carbohydrate-binding properties are tightly related to its antifungal activity; these are the first insights into the molecular basis of the functioning of DUF26 protein family members.     

A novel cluster of mariner-like elements belonging to mellifera subfamily from spiders and insects: implications of recent horizontal transfer on the South-West Islands of Japan

Mariner-like elements (MLEs) have been isolated from various eukaryotic genomes and they are divided into 15 subfamilies, including main five subfamilies: mauritiana, cecropia, mellifera/capitata, irritans, and elegans/briggsae. In the present study, MLEs belonging to mellifera subfamily were isolated from various spiders and insects (Hymenoptera and Lepidoptera) inhabiting the South-West Islands of Japan and neighboring regions. MLEs isolated from 15 different species formed a distinct novel cluster in mellifera subfamily. MLEs obtained from three different species [i.e., the bee Amegilla senahai subflavescens (Amsmar1), the wasp Campsomeris sp. (Casmar1), and the swallowtail butterfly Pachliopta aristolochiae (Paamar1)] contained an intact open reading frame that encoded a putative transposase. These transposases exhibited high similarity of 97.9 % among themselves. In case of Casmar1, the presence of an intact ORF was found in high frequencies (i.e., 11 out of 12 clones). In addition, these transposases also showed the presence of a terminal inverted repeat-binding motif, DD(34)D and two highly conserved amino acid motifs, (W/L)(I/L)PHQL and YSP(D/N)L(A/S)P. These two motifs differed from previously known motifs, WVPHEL and YSPDLAP. MLEs isolated from these three different species may have been inserted into their genomes by horizontal transfer. Furthermore, the presence of an intact ORF suggests that they are still active in habitats along these isolated islands.