The glycosphingolipid sulfatide in the islets of Langerhans in rat pancreas is processed through recycling: possible involvement in insulin trafficking

In previous studies we have shown that sulfatide (galactosylceramide-3-O-sulfate), in various species, is present in the insulin-producing cells in pancreatic islets of Langerhans. In this study the synthesis of sulfatide in the islets has been investigated by pulse chase labeling at varying glucose levels and in the presence or absence of the glycosphingolipid synthesis inhibitory agents, Brefeldin A, fumonisin B1 and chloroquine and the distribution of sulfatide by immune-electronmicroscopy. The data showed that (1) sulfatide was produced in islets of Langerhans, (2) the main pathway for synthesis was through recycling involving partial degradation in the lysosome, and that (3) high glucose levels, although not primarily reflected in an increased synthesis of sulfatide, lead to an increased expression of mRNA for the UDP-galactose:ceramide galactosyltransferase, producing the immediate precursor of sulfatide. Furthermore, mass spectrometry analyses revealed a high proportion of short chain fatty acids, C16:0 (50%) and no hydroxylated forms and thus special physicochemical properties, indicating important differences between pancreatic and brain/neural sulfatide. Immune electron microscopy revealed an intracellular expression of sulfatide in the secretory granules, the Golgi network and the lysosomes of the islets. These results indicate that sulfatide follows the same intracellular route as insulin and suggest a functional association between these molecules. We have raised the hypothesis that sulfatide possibly plays a role in the trafficking of insulin in the islets of Langerhans in rat pancreas.     

A novel disorder caused by defective biosynthesis of N-linked oligosaccharides due to glucosidase I deficiency

Glucosidase I is an important enzyme in N-linked glycoprotein processing, removing specifically distal alpha-1,2-linked glucose from the Glc3Man9GlcNAc2 precursor after its en bloc transfer from dolichyl diphosphate to a nascent polypeptide chain in the endoplasmic reticulum. We have identified a glucosidase I defect in a neonate with severe generalized hypotonia and dysmorphic features. The clinical course was progressive and was characterized by the occurrence of hepatomegaly, hypoventilation, feeding problems, seizures, and fatal outcome at age 74 d. The accumulation of the tetrasaccharide Glc(alpha1-2)Glc(alpha1-3)Glc(alpha1-3)Man in the patient's urine indicated a glycosylation disorder. Enzymological studies on liver tissue and cultured skin fibroblasts revealed a severe glucosidase I deficiency. The residual activity was <3% of that of controls. Glucosidase I activities in cultured skin fibroblasts from both parents were found to be 50% of those of controls. Tissues from the patient subjected to SDS-PAGE followed by immunoblotting revealed strongly decreased amounts of glucosidase I protein in the homogenate of the liver, and a less-severe decrease in cultured skin fibroblasts. Molecular studies showed that the patient was a compound heterozygote for two missense mutations in the glucosidase I gene: (1) one allele harbored a G-->C transition at nucleotide (nt) 1587, resulting in the substitution of Arg at position 486 by Thr (R486T), and (2) on the other allele a T-->C transition at nt 2085 resulted in the substitution of Phe at position 652 by Leu (F652L). The mother was heterozygous for the G-->C transition, whereas the father was heterozygous for the T-->C transition. These base changes were not seen in 100 control DNA samples. A causal relationship between the alpha-glucosidase I deficiency and the disease is postulated.     

Human C4b-binding protein has overlapping, but not identical, binding sites for C4b and streptococcal M proteins

Many strains of Streptococcus pyogenes bind C4b-binding protein (C4BP), an inhibitor of complement activation. The binding is mediated by surface M proteins in a fashion that has been suggested to mimic the binding of C4b. We have previously shown that a positively charged cluster at the interface between complement control protein domains 1 and 2 of C4BP alpha-chain is crucial for the C4b-C4BP interaction. To extend this observation, and to investigate the interaction with M proteins, we constructed and characterized a total of nine mutants of C4BP. We identified a key recognition surface for M proteins that overlaps with the C4b binding site because substitution of R64 and H67 by Gln dramatically reduces binding to both ligands. However, the analysis of all mutants indicates that the binding sites for C4b and M proteins are only overlapping, but not identical. Furthermore, M proteins were able to displace C4BP from immobilized C4b, whereas C4b only weakly affected binding of C4BP to immobilized M proteins. We found that the molecular mechanisms involved in these two interactions differ because the binding between M proteins and C4BP is relatively insensitive to salt in contrast to the C4BP-C4b binding. In addition, six mAbs directed against the alpha-chain interfered with C4b-C4BP interaction, whereas only two of them efficiently inhibited binding of C4BP to M proteins. Collectively, our results suggest that binding between C4b and C4BP is governed mostly by electrostatic interactions, while additional noncovalent forces cause tight binding of C4BP to streptococcal M proteins.     

The human tRNA(m(2)(2)G(26))dimethyltransferase: functional expression and characterization of a cloned hTRM1 gene

This paper presents the first example of a complete gene sequence coding for and expressing a biologically functional human tRNA methyltransferase: the hTRM1 gene product tRNA(m²₂G)dimethyltransferase. We isolated a human cDNA (1980 bp) made from placental mRNA coding for the full-length (659 amino acids) human TRM1 polypeptide. The sequence was fairly similar to Saccharomyces cerevisiae Trm1p, to Caenorhabditis elegans TRM1p and to open reading frames (ORFs) found in mouse and a plant (Arabidopsis thaliana) DNA. The human TRM1 gene was expressed at low temperature in Escherichia coli as a functional recombinant protein, able to catalyze the formation of dimethylguanosine in E.coli tRNA in vivo. It targeted solely position G₂₆ in T7 transcribed spliced and unspliced human tRNA^Tyr in vitro and in yeast trm1 mutant tRNA. Thus, the human TRM1 protein is a tRNA(m²₂G₂₆)dimethyltransferase. Compared with yeast Trm1p, hTRM1p has a C-terminal protrusion of ~90 amino acids which shows similarities to a mouse protein related to RNA splicing. A deletion of these 90 C-terminal amino acids left the modification activity in vitro intact. Among point mutations in the hTRM1 gene, only those located in conserved regions of hTRM1p completely eliminated modification activity.     

Differential expression of four genes encoding 1-aminocyclopropane-1-carboxylate synthase in Lupinus albus during germination, and in response to indole-3-acetic acid and wounding

1-Aminocyclopropane-1-carboxylate (ACC) synthase (ACS; EC 4.4.1.14) is the key regulatory enzyme of the ethylene biosynthetic pathway and is encoded by a multigene family in Arabidopsis thaliana, tomato, mung bean and other plants. Southern blot analysis revealed the existence of at least five ACS genes in white lupin (Lupinus albus L.) genome. Four complete and one partial sequences representing different ACS genes were cloned from the lupin genomic library. The levels of expression of two of the genes, LA-ACS1 and LA-ACS3, were found to increase after hypocotyl wounding. Apparently, these two genes were up-regulated by exogenous IAA treatment of seedlings. The LA-ACS3 mRNA levels were also elevated in the apical part of hypocotyl, which is reported to contain a high endogenous auxin concentration. This gene may be involved in the auxin- and ethylene-controlled apical hook formation. The expression of the LA-ACS4 gene was found to be almost undetectable. This gene may represent a “silent” twin of LA-ACS5 as these two genes share a considerable level of homology in coding and non-coding regions. The LA-ACS5 mRNA is strongly up-regulated in the embryonic axis of germinating seeds at the time of radicle emergence, and was also found in roots and hypocotyls of lupin seedlings. Received: 19 July 1999 / Accepted: 3 March 2000     

Cytological basis for a tetraspory in Cupressus sempervirens L. megagametogenesis and its implications in genetic studies

 The processes of megasporogenesis and early megagametogenesis were cytologically investigated in Cupressus sempervirens L. in order to elucidate, at the cellular level, the origin of the megagametophyte. After pollination, sporogenous tissue developed in the chalazal region of the nucellus, but only one megaspore mother cell differentiated and divided meiotically without cell-wall formation. This led to the development of a cell with four nuclei which directly functioned as a megaspore. The C. sempervirens megagametophyte is thus tetrasporic, in contrast to the majority of conifers where the megagametophyte is monosporic. The consequenses of this observation are discussed from a genetics point of view. Received: 15 August 1997 / Accepted: 19 September 1997     

Rapid regeneration of whole plants in large crabgrass (Digitaria sanguinalis L.) using thin-cell-layer culture

A method was designed to produce rapidly (10–14 days) and directly (without intermediate callus) whole plants of Digitaria sanguinalis with a high yield without subculture. These plants developed from new structures, designated “pseudo-embryogenic structures”, initiated only 1 week after the culture of tranverse thin cell layers (tTCLs), i.e., thin stem sections, on a Murashige and Skoog medium containing 3% sucrose and a combination of a low concentration of 2,4-dichlorophenoxyacetic acid (1 μm) and a high concentration of 6-benzylaminopurine (10 μm). The fresh weight of plants regenerated per tTCL on gelrite was 6 times higher than with agar and 30 times higher than with agarose. Received: 25 August 1997 / Revision received: 18 February 1998 / Accepted: 10 March 1998     

Characterization of terminal NeuNAcalpha2-3Galbeta1-4GlcNAc sequence in lipooligosaccharides of Neisseria meningitidis

Group B and C Neisseria meningitidis are the major cause of meningococcal disease in the United States and in Europe. N . meningitidis lipooligosaccharide (LOS), a major surface antigen, can be divided into 12 immunotypes of which L1 through L8 were found among Group B and C organisms. Groups B and C but not Group A may sialylate their LOSs with N-acetylneuraminic acid (NeuNAc) at the nonreducing end because they synthesize CMP-NeuNAc. Using sialic acid-galactose binding lectins as probes in an ELISA format, six of the eight LOS immunotypes (L2, L3, L4, L5, L7, and L8) in Groups B and C bound specifically to Maackia amurensis leukoagglutinin (MAL), which recognizes NeuNAcalpha2- 3Galbeta1-4GlcNAc/Glc sequence, but not to Sambucus nigra agglutinin, which binds NeuNAcalpha2-6Gal sequence. The combination of SDS-PAGE and MAL-blot analyses revealed that these six LOSs contained only the NeuNAcalpha2-3Galbeta1-4GlcNAc trisaccharide sequence in their 4.1 kDa LOS components, which have a common terminal lacto-N-neotetraose (LNnT, Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc) structure when nonsialylated as shown by previous studies. The LOS-lectin binding was abolished when the LOSs were treated with Newcastle disease viral neuraminidase which cleaves alpha2-->3 linked sialic acid. Methylation analysis of a representative LOS (L2) confirmed that NeuNAc is 2-->3 linked to Gal. Thus, these LOSs structurally mimic certain glycolipids, i.e., paragloboside (LNnT-ceramide) and sialylparagloboside and some glycoproteins in having LNnT and N-acetyllactosamine sequences, respectively, with or without alpha2-->3 linked NeuNAc. The molecular mimicry of the LOSs may play a role in the pathogenesis of N.meningitidis by assisting the organism to evade host immune defenses in man.      

A web server to locate periodicities in a sequence

Summary : FT is a tool written in C++, which implements the Fourier analysis method to locate periodicities in aminoacid or DNA sequences. It is provided for free public use on a WWW server with a Java interface. Availability : The server address is http://o2.db. uoa.gr/FT Contact : shamodr@atlas.uoa.gr