Characterization of the lacto series of gangliosides, especially of disialo-lacto-N-norhexaosyl ceramide isolated from adult bovine nasal cartilage

A disialosylganglioside was isolated from adult bovine nasal cartilage, and its structure was determined by analysis of sugar composition, permethylation analysis, exoglycosidase treatment, and mild acid hydrolysis. The structure of this ganglioside was identified as disialo-lacto-N-norhexaosyl ceramide, NeuNAc(alpha 2-8)NeuNAc-(alpha 2-3)Gal(beta 1-4)GlcNAc(beta 1-3)Gal(beta 1-4)GlcNAc(beta 1-3)Gal(1-4)Glc(1-1)Cer. Furthermore, we also isolated from this cartilage gangliosides whose structures were presumed to be monosialo-lacto-N-norhexaosyl ceramide, and mono- and disialo-lacto-N-neotetraosyl ceramide. The major fatty acids of the four gangliosides isolated were palmitic, stearic, behenic and lignoceric acids. The predominant long chain bases were sphingenine, heptadecasphingenine and hexadecasphingenine.     

EphB receptors trigger Akt activation and suppress Fas receptor-induced apoptosis in malignant T lymphocytes

Treatment of hematopoietic malignancies often requires allogeneic bone marrow transplantation, and the subsequent graft-versus-leukemia response is crucial for the elimination of malignant cells. Cytotoxic T lymphocytes and NK cells responsible for the immunoelimination express Fas ligand and strongly rely on the induction of Fas receptor-mediated apoptosis for their action. Although cancer cells are removed successfully by graft-versus-leukemia reactions in myeloid malignancies, their efficiency is low in T cell leukemias. This may be partially because of the ability of malignant T cells to escape apoptosis. Our work shows that Eph family receptor EphB3 is consistently expressed by malignant T lymphocytes, most frequently in combination with EphB6, and that stimulation with their common ligands, ephrin-B1 and ephrin-B2, strongly suppresses Fas-induced apoptosis in these cells. This effect is associated with Akt activation and with the inhibition of the Fas receptor-initiated caspase proteolytic cascade. Akt proved to be crucial for the prosurvival response, because inhibition of Akt, but not of other molecules central to T cell biology, including Src kinases, MEK1 and MEK2, blocked the antiapoptotic effect. Overall, this demonstrates a new role for EphB receptors in the protection of malignant T cells from Fas-induced apoptosis through Akt engagement and prevention of caspase activation. Because Fas-triggered apoptosis is actively involved in the graft-versus-leukemia response and cytotoxic T cells express ephrin-Bs, our observations suggest that EphB receptors are likely to support immunoevasivenes of T cell malignancies and may represent promising targets for therapies, aiming to enhance immunoelimination of cancerous T cells.     

Can plants serve as a vector for prions causing chronic wasting disease?

Prions, the causative agent of chronic wasting disease (CWD) enter the environment through shedding of bodily fluids and carcass decay, posing a disease risk as a result of their environmental persistence. Plants have the ability to take up large organic particles, including whole proteins, and microbes. This study used wheat (Triticum aestivum L.) to investigate the uptake of infectious CWD prions into roots and their transport into aerial tissues. The roots of intact wheat plants were exposed to infectious prions (PrP^TSE) for 24 h in three replicate studies with PrP^TSE in protein extracts being detected by western blot, IDEXX and Bio-Rad diagnostic tests. Recombinant prion protein (PrP^C) bound to roots, but was not detected in the stem or leaves. Protease-digested CWD prions (PrP^TSE) in elk brain homogenate interacted with root tissue, but were not detected in the stem. This suggests wheat was unable to transport sufficient PrP^TSE from the roots to the stem to be detectable by the methods employed. Undigested PrP^TSE did not associate with roots. The present study suggests that if prions are transported from the roots to the stems it is at levels that are below those that are detectable by western blot, IDEXX or Bio-Rad diagnostic kits.     

Comprehensive sequence analysis of translation termination sites in various eukaryotes

Recent investigations into the translation termination sites of various organisms have revealed that not only stop codons but also sequences around stop codons have an effect on translation termination. To investigate the relationship between these sequence patterns and translation as well as its termination efficiency, we analysed the correlation between strength of consensus and translation efficiency, as predicted according to Codon Adaptation Index (CAI) value. We used RIKEN full-length mouse cDNA sequences and ten other eukaryotic UniGene datasets from NCBI for the analyses. First, we conducted sequence profile analyses following translation termination sites. We found base G and A at position +1 as a strong consensus for mouse cDNA. A similar consensus was found for other mammals, such as Homo sapiens, Rattus norvegicus and Bos taurus. However, some plants had different consensus sequences. We then analysed the correlation between the strength of consensus at each position and the codon biases of whole coding regions, using information content and CAI value. The results showed that in mouse cDNA, CAI value had a positive correlation with information content at positions +1. We also found that, for positions with strong consensus, the strength of the consensus is likely to have a positive correlation with CAI value in some other eukaryotes. Along with these observations, biological insights into the relationship between gene expression level, codon biases and consensus sequence around stop codons will be discussed.     

Calcium oscillations induced by gambierol in cerebellar granule cells

Gambierol is a marine polyether ladder toxin derived from the dinoflagellate Gambierdiscus toxicus. To date, gambierol has been reported to act either as a partial agonist or as an antagonist of sodium channels or as a blocker of voltage‐dependent potassium channels. In this work, we examined the cellular effect of gambierol on cytosolic calcium concentration, membrane potential and sodium and potassium membrane currents in primary cultures of cerebellar granule cells. We found that at concentrations ranging from 0.1 to 30 µM, gambierol‐evoked [Ca²⁺]c oscillations that were dependent on the presence of extracellular calcium, irreversible and highly synchronous. Gambierol‐evoked [Ca²⁺]c oscillations were completely eliminated by the NMDA receptor antagonist APV and by riluzole and delayed by CNQX. In addition, the K⁺ channel blocker 4‐aminopyridine (4‐AP)‐evoked cytosolic calcium oscillations in this neuronal system that were blocked by APV and delayed in the presence of CNQX. Electrophysiological recordings indicated that gambierol caused membrane potential oscillations, decreased inward sodium current amplitude and decreased also outward IA and IK current amplitude. The results presented here point to a common mechanism of action for gambierol and 4‐AP and indicate that gambierol‐induced oscillations in cerebellar neurons are most likely secondary to a blocking action of the toxin on voltage‐dependent potassium channels and hyperpolarization of sodium current activation. J. Cell. Biochem. 110: 497–508, 2010. © 2010 Wiley‐Liss, Inc.     

Characteristics of β-galactosidase purified from cell suspension cultures of carrot

Five glycosidase activities from cell homogenate of carrot ( Daucus carota L. cv. Kintoki) cell cultures were assayed after extraction successively by phosphate buffer (pH 7.0) and the buffer plus 2 M NaCl. A β-galactosidase (EC 3.2.1.23) was isolated in a highly purified state from the buffer-soluble protein fraction by ammonium sulfate fractionation and chromatography on CM-Sephadex C-50, DEAE-Sephadex A-50 and Sephadex G-200. The molecular weight of this enzyme was ca 104 000 and the isoelectric point was pH 7.8. The optimal activity occurred at pH 4.4 with McIlvaine buffer. The K_m and V_max values were 1.67 m M and 201 units (mg protein)⁻¹, respectively, for p -nitrophenyl β- d -galactopyranoside. The enzyme activity was strongly inhibited by Zn²⁺, Cu²⁺, Hg²⁺ and d -galactono-1,4-lactone. The enzyme acted on the β-1,4-linked galactan prepared from citrus pectin in an exo-fashion. Furthermore, the enzyme was slightly involved in the hydrolysis of the pectic polymer and cell walls purified from carrot cell cultures.     

Two distinct cDNAs for human IMP dehydrogenase

IMP dehydrogenase (EC 1.1.1.205), the rate-limiting enzyme of de novo GTP biosynthesis, is a promising target in antileukemic chemotherapy. We have isolated two distinct cDNA clones (types I and II) encoding IMP dehydrogenase from a human spleen cDNA library. Both clones encode closely related proteins of 514 residues showing 84% sequence identity. Northern hybridization analyses of poly(A)+ RNA from human normal leukocytes and human ovarian tumors demonstrated a striking contrast in mRNA expression in that type I mRNA is the main species in normal leukocytes and type II predominates over type I in the tumor. This is the first report suggesting the existence of two distinct types of human IMP dehydrogenase molecular species which may have different sensitivities to the drugs targeted against IMP dehydrogenase.     

Effect of the recombinant vaccinia viruses that express HTLV-I envelope gene on HTLV-I infection.

The human T-lymphotropic virus type I (HTLV-I) is etiologically linked to adult T-cell leukemia (ATL). To develop a vaccine against ATL, we constructed recombinant vaccinia viruses containing the envelope gene of HTLV-I in the vaccinia virus hemagglutinin (HA) gene, a new site where foreign genes can be inserted. A single inoculation of the recombinant virus induced antibodies to the env proteins of HTLV-I in rabbits and had a protective effect against HTLV-I infection.     

Studies on the Pectic Depolymerases I. Exopolygalacturonase from Root Tissues and Cell Suspension Cultures of Carrots

Three polygalacturonases (PG) have been isolated from carrots(Daucus carota L. cv. Kintoki). Two were isolated from roottissues (PG-I and PG-II) and one from cell suspension cultures(PG-III). PG-I and PG-III were readily solubilized in a lowionic strength buffer, whereas PG-II required additional NaClto be solubilized. These seems to be a change in the propertiesof PG between the original tissue and carrot cell cultures.The three PGs were partially purified by chromatography on SephadexG-150, and characterized. Elution from a Sephadex G-150 column indicated a molecular weightof about 48,000 for all three PGs. PG-III, studied in detail,hydrolyzed the galacturonan chain in an exo-fashion, and wasnot activated by a variety of cations at concentrations of 0.5or 1.0 mM. The pH optimum, and pH and heat stability of PG-Iand PG-III were slightly different from those of PG-II. PG-Iwas also different from PG-II and PG-III in its pectin hydrolyzingactivity. These results indicate that the enzymatic properties of PG-IIIfrom cell cultures are very similar to those of PG-I or PG-IIfrom root tissues; the only significant difference seems tobe the binding properties of the PGs to the cell wall materials. (Received March 23, 1981; Accepted June 11, 1981)