Effect of exo-β-D-(1→3)-galactanase digestion on complement activating activity of neutral arabinogalactan unit in a pectic arabinogalactan from roots of Angelica acutiloba Kitagawa

A complement activating pectic arabinogalactan (AGIIb-1) has been isolated from roots of Angelica acutiloba Kitagawa, and a neutral arabinogalactan (N-I) unit, which was liberated from a side chain in AGIIb-1 by mild acid hydrolysis, has found to show the most potent complement activating activity among the arabinogalactan side chains of AGIIb-1. In order to clarify the essential carbohydrate side chains in N-I for the activity, neutral arabinogalactan (AF-N-I) unit, which was obtained from exo--L-arabinofuranosidase-digested AGIIb-1, was digested with exo-β-d-(1 → 3)-galactanase from Irpex lacteus. The galactanase digestion of the AF-N-I unit completely stopped its reactivity with β-d-glucosyl-Yariv antigen, and significantly reduced its complement activating activity. The digestion products gave high molecular weight fragments (GN-1A and -1B) and intermediate size fragments in addition to β-d-(1 → 6)-galactosyl mono to pentasaccharides as the side chains of the AF-N-I unit. Among the fragments, GN-1A, GN-1B and intermediate size fragments showed relatively potent complement activating activity, suggesting that these side chains in the N-I unit might be responsible for expression of the activity of the N-I unit.     

Binding of metal ions toE. coli RNase HI observed by1H−15N heteronuclear 2D NMR

Summary The divalent metal ion binding site and binding constant of ribonuclease HI fromEscherichia coli were investigated by observing chemical shift changes using¹H–¹⁵N heteronuclear NMR. Chemical shift changes were monitored during the titration of the enzyme with salts of the divalent cations. The enzyme was uniformly labeled by¹⁵N, which facilitated the monitoring of the chemical shift change of each cross peak between the backbone amide proton and the amide¹⁵N. The chemical shifts of several amide groups were affected upon the addition of a divalent metal ion: Mg²⁺, Ca²⁺, or Ba²⁺. These amide groups resided close to the active site, consistent with the previous X-ray crystallographic studies. From the titration analysis, a single divalent ion binding site was observed with a weak binding constant (K_D=2–4 mM for the current divalent ions).     

Nonprotein amino acid furanomycin, unlike isoleucine in chemical structure, is charged to isoleucine tRNA by isoleucyl-tRNA synthetase and incorporated into protein

Nonprotein amino acid furanomycin was found to bind with Escherichia coli isoleucyl-tRNA synthetase (IleRS) almost as tightly as the substrate L-isoleucine. The conformation of furanomycin bound to the enzyme was determined by NMR analyses including the transferred nuclear Overhauser effect method. The conformation of IleRS-bound furanomycin was similar to that of L-isoleucine, although the chemical structure of furanomycin is unlike that of L-isoleucine. By E. coli IleRS, E. coli tRNAIle was charged with furanomycin as efficiently as with L-isoleucine. Furthermore, furanomycyl-tRNAIle was bound to polypeptide chain elongation factor Tu as tightly as isoleucyl-tRNAIle. Furanomycin was found to be incorporated into beta-lactamase precursor by in vitro protein biosynthesis. A newly designed amino acid will probably be incorporated into proteins, provided that the new amino acid takes a similar conformation as a protein-constituting amino acid in the active site of an aminoacyl-tRNA synthetase.     

Localization and expression of AQP5 in cornea, serous salivary glands, and pulmonary epithelial cells

Aquaporin (AQP) 5 gene was recently isolated from salivary glandand identified as a member of the AQP family. The mRNAexpression and localization have been examined in several organs. Thepresent study was focused on elucidation of AQP5 expression andlocalization in the eye, salivary gland, and lung in rat. RNaseprotection assay confirmed intense expression of AQP5 mRNA in theseorgans but negligible expression in other organs. To examine the mRNA expression sites in the eye, several portions were microdissected fortotal RNA isolation. AQP5 mRNA was enriched in cornea but not in otherportions (retina, lens, iris/ciliary body, conjunctiva, or sclera).AQP5 was selectively localized on the surface of corneal epithelium inthe eye by immunohistochemistry and immunoelectron microscopy using anaffinity-purified anti-AQP5 antibody. AQP5 was also localized on apicalmembranes of acinar cells in the lacrimal gland and on the microvilliprotruding into intracellular secretory canaliculi of the seroussalivary gland. In the lung, apical membranes of type I pulmonaryepithelial cells were also immunostained with the antibody. Thesefindings suggest a role of AQP5 in water transport to preventdehydration or to secrete watery products in these tissues.

     

Notes on new Agaricales of Japan 1

Three new species of Agaricales are described and illustrated from eastern Honshu, Japan:Collybia effusa sp. nov. (sectionLevipedes), forming a distinctly sulcate-striate pileus and distant lamellae, was found on dead twigs ofCryptomeria japonica andCallicarpa japonica; Collybia macrosperma sp. nov., forming cylindrical-fusoid, relatively large-sized basidiospores and whitish basidiomata densely covered with fine, soft hairs, was found on dead fallen twigs in aCarpinus andQuercus forest:Marasmiellus, gregarius sp. nov (subsectionRamealini of sectionRameales), forming small, pale colored pilei and diverticulate cheilocystidia, was found on dead twigs ofHydrangea involucrata andTrachelospermum asiaticum.     

Gene transfer of PEDF attenuates ischemic brain damage in the rat middle cerebral artery occlusion model

Pigment epithelium-derived factor (PEDF) is a 50-kDa glycoprotein that protects various types of cultured neurons against neurotoxic stimuli, but its precise role in the CNS is not fully understood. In this study, we used rats whose brains were transfected to over-express human PEDF in order to elucidate the neuroprotective effect of PEDF following transient middle cerebral artery occlusion (MCAO). A replication-defective adenoviral vector containing the human PEDF gene (Ad.PEDF) or E. coli β-galactosidase (Ad.LacZ) was directly injected into the right striatum at 7 days prior to 70 min of MCAO in rats. Infarct volume and degree of edema of the Ad.PEDF-treated group were significantly reduced compared to the Ad.LacZ-treated group 24 h after MCAO. Degeneration of neurons, astrocytes, and oligodendrocytes caused by MCAO were attenuated by over-expression of PEDF. The up-regulation of pro-inflammatory genes (TNFα, IL-1β, IL-6, COX-2, and iNOS) and water channel aquaporin 4 after MCAO was significantly reduced in Ad.PEDF-injected striatum. In conclusion, the results from this study provide the first in vivo evidence that PEDF is effective in protecting CNS neurons from ischemic insult, suggesting that PEDF may have a role as an endogenous neuroprotectant in the CNS.     

Variation in the geometry of foreleg claws in sympatric giant water bug species: an adaptive trait for catching prey?

When giant water bugs (Heteroptera: Belostomatidae) encounter prey animals that are larger than they are themselves, they first hook the claw of their raptorial legs onto the animal, and then use all their legs to pin it. The claws of the raptorial legs in giant water bugs play an important role in catching larger prey, but the relationship between the claws, body lengths of predators, and prey size has not been fully investigated. To elucidate the functioning of claws in catching prey, we investigated prey body size relative to predator size in nymphs of two sympatric belostomatid giant water bug species, the vertebrate eater Kirkaldyia (=Lethocerus) deyrolli Vuillefroy and the invertebrate eater Appasus japonicus Vuillefroy, captured in rice fields. The younger nymphs of K. deyrolli caught preys that were larger than themselves, whereas those of A. japonicus caught preys that were smaller. Younger nymphs of K. deyrolli had claws that were curved more sharply than those of A. japonicus. The more curved claws of younger nymphs of K. deyrolli probably hook more easily onto larger vertebrates and thus this shape represents an adaptation for acquiring such prey.     

Anterior pituitary progenitor cells express costimulatory molecule 4Ig-B7-H3

Stem/Progenitor cells in the postnatal pituitary gland are embedded in a marginal cell layer around Rathke's pouch. However, the nature and behavior of anterior pituitary progenitor cells remain unclear. We established bovine anterior pituitary progenitor cell line (BAPC)-1 from the anterior pituitary gland, which expressed stem/progenitor cell-related genes and several inflammatory cytokines. To characterize and localize these pituitary progenitor cells, we produced a mAb (12B mAb) against BAPC-1. The 12B mAb recognized the 4Ig-B7-H3 molecule, which is a costimulatory molecule and negative regulator in T cell activation. WC1(+) gammadelta T cells in young bovine PBMC express the 4Ig-B7-H3 molecule, but few or no 4Ig-B7-H3-immunoreactive cells are expressed in PBMC in adult cattle. The 12B-immunoreactive cells in the bovine anterior pituitary gland were localized around Rathke's pouch and expressed IL-18 and MHC class II. However, the number of 12B-immunoreactive cells was lower in adult than in young cattle. BAPC-1 expressed IL-18 and MHC class II, and demonstrated phagocytotic activity. BAPC-1 also had the ability to promote CD25 expression in PBMC after 5 days of coculture, and blocking 4Ig-B7-H3 x 12B mAb enhanced their expression of CD25. In addition, the 12B-immunoreactive cells were observed around the pars tuberalis closely bordering the median eminence and in the blood vessels of the primary portal plexus in the anterior pituitary gland. These results suggest that an established BAPC-1 may originate from these progenitor cells, and that the progenitor cells with 4Ig-B7-H3 may play a critical role in the immunoendocrine network.     

Dynamic morphologies of pollen plastids visualised by vegetative-specific FtsZ1–GFP in Arabidopsis thaliana

The behaviour and multiplication of pollen plastids have remained elusive despite their crucial involvement in cytoplasmic inheritance. Here, we present live images of plastids in pollen grains and growing tubes from transgenic Arabidopsis thaliana lines expressing stroma-localised FtsZ1–green-fluorescent protein fusion in a vegetative cell-specific manner. Vegetative cells in mature pollen contained a morphologically heterogeneous population of round to ellipsoidal plastids, whilst those in late-developing (maturing) pollen included plastids that could have one or two constriction sites. Furthermore, plastids in pollen tubes exhibited remarkable tubulation, stromule (stroma-filled tubule) extension, and back-and-forth movement along the direction of tube growth. Plastid division, which involves the FtsZ1 ring, was rarely observed in mature pollen grains.