Isolation of EST-derived microsatellite markers for genotyping the A and B genomes of wheat

Genetic variation present in 64 durum wheat accessions was investigated by using three sources of microsatellite (SSR) markers: EST-derived SSRs (EST-SSRs) and two sources of SSRs isolated from total genomic DNA. Out of 245 SSR primer pairs screened, 22 EST-SSRs and 20 genomic-derived SSRs were polymorphic and used for genotyping. The EST-SSR primers produced high quality markers, but had the lowest level of polymorphism (25%) compared to the other two sources of genomic SSR markers (53%). The 42 SSR markers detected 189 polymorphic alleles with an average number of 4.5 alleles per locus. The coefficient of similarity ranged from 0.28 to 0.70 and the estimates of similarity varied when different sources of SSR markers were used to genotype the accessions. This study showed that EST-derived SSR markers developed in bread wheat are polymorphic in durum wheat when assaying loci of the A and B genomes. A minumum of ten EST-SSRs generated a very low probability of identity (0.36×10⁻¹²) indicating that these SSRs have a very high discriminatory power. EST-SSR markers directly sample variation in transcribed regions of the genome, which may enhance their value in marker-assisted selection, comparative genetic analysis and for exploiting wheat genetic resources by providing a more-direct estimate of functional diversity. Received: 19 December 2000 / Accepted: 17 April 2001     

Lhca5 interaction with plant photosystem I

In the outer antenna (LHCI) of higher plant photosystem I (PSI) four abundantly expressed light-harvesting protein of photosystem I (Lhca)-type proteins are organized in two heterodimeric domains (Lhca1/Lhca4 and Lhca2/Lhca3). Our cross-linking studies on PSI-LHCI preparations from wildtype Arabidopsis and pea plants indicate an exclusive interaction of the rarely expressed Lhca5 light-harvesting protein with LHCI in the Lhca2/Lhca3-site. In PSI particles with an altered LHCI composition Lhca5 assembles in the Lhca1/Lhca4 site, partly as a homodimer. This flexibility indicates a binding-competitive model for the LHCI assembly in plants regulated by molecular interactions of the Lhca proteins with the PSI core.     

Cutting edge: Deficiency of macrophage migration inhibitory factor impairs murine airway allergic responses

Increased levels of macrophage migration inhibitory factor (MIF) in serum, sputum, and bronchioalveolar lavage fluid (BALF) from asthmatic patients and time/dose-dependent expression of MIF in eosinophils in response to phorbol myristate acetate suggest the participation of MIF in airway inflammation. In this study, we examined inflammation in OVA-sensitized mouse lungs in wild-type and MIF-deficient mice (MIF(-/-)). We report increased MIF in the lung and BALF of sensitized wild-type mice. MIF(-/-) mice demonstrated significant reductions in serum IgE and alveolar inflammatory cell recruitment. Reduced Th1/Th2 cytokines and chemokines also were detected in serum or BALF from MIF(-/-) mice. Importantly, alveolar macrophages and mast cells, but not dendritic cells or splenocytes, from MIF(-/-) mice demonstrated impaired CD4+ T cell activation, and the reconstitution of wild-type mast cells in MIF(-/-) mice restored the phenotype of OVA-induced airway inflammation, revealing a novel and essential role of mast cell-derived MIF in experimentally induced airway allergic diseases.     

Kidney sulfatides in mouse models of inherited glycosphingolipid disorders: determination by nano-electrospray ionization tandem mass spectrometry

Sulfatides show structural, and possibly physiological similarities to gangliosides. Kidney dysfunction might be correlated with changes in sulfatides, the major acidic glycosphingolipids in this organ. To elucidate their in vivo metabolic pathway these compounds were analyzed in mice afflicted with inherited glycosphingolipid disorders. The mice under study lacked the genes encoding either beta-hexosaminidase alpha-subunit (Hexa-/-), the beta-hexosaminidase beta-subunit (Hexb-/-), both beta-hexosaminidase alpha and beta-subunits (Hexa-/- and Hexb-/-), GD3 synthase (GD3S-/-), GD3 synthase and GalNAc transferase (GD3S-/- and GalNAcT-/-), GM2 activator protein (Gm2a-/-), or arylsulfatase A (ASA-/-). Quantification of the sulfatides, I(3)SO(3)(-)-GalCer (SM4s), II(3)SO(3)(-)-LacCer (SM3), II(3)SO(3)(-)-Gg(3)Cer (SM2a), and IV(3,) II(3)-(SO(3)(-))(2)-Gg(4)Cer (SB1a), was performed by nano-electrospray tandem mass spectrometry. We conclude for the in vivo situation in mouse kidneys that: 1) a single enzyme (GalNAc transferase) is responsible for the synthesis of SM2a and GM2 from SM3 and GM3, respectively. 2) In analogy to GD1a, SB1a is degraded via SM2a. 3) SM2a is hydrolyzed to SM3 by beta-hexosaminidase S (Hex S) and Hex A, but not Hex B. Both enzymes are supported by GM2-activator protein. 4) Arylsulfatase A is required to degrade SB1a. It is probably the sole sphingolipid-sulfatase cleaving the galactosyl-3-sulfate bond. In addition, a human Tay-Sachs patient's liver was investigated, which showed accumulation of SM2a along with GM2 storage. The different ceramide compositions of both compounds indicated they were probably derived from different cell types. These data demonstrate that in vivo the sulfatides of the ganglio-series follow the same metabolic pathways as the gangliosides with the replacement of sulfotransferases and sulfatases by sialyltransferases and sialidases. Furthermore, a novel neutral GSL, IV(6)GlcNAcbeta-Gb(4)Cer, was found to accumulate only in Hexa-/- and Hexb-/- mouse kidneys. From this we conclude that Hex S also efficiently cleaves terminal beta1-6-linked HexNAc residues from neutral GSLs in vivo.     

X-ray crystallographic structures of the Escherichia coli periplasmic protein FhuD bound to hydroxamate-type siderophores and the antibiotic albomycin.

Siderophore-binding proteins play an essential role in the uptake of iron in many Gram-positive and Gram-negative bacteria. FhuD is an ATP-binding cassette-type (ABC-type) binding protein involved in the uptake of hydroxamate-type siderophores in Escherichia coli. Structures of FhuD complexed with the antibiotic albomycin, the fungal siderophore coprogen and the drug Desferal have been determined at high resolution by x-ray crystallography. FhuD has an unusual bilobal structure for a periplasmic ligand binding protein, with two mixed beta/alpha domains connected by a long alpha-helix. The binding site for hydroxamate-type ligands is composed of a shallow pocket that lies between these two domains. Recognition of siderophores primarily occurs through interactions between the iron-hydroxamate centers of each siderophore and the side chains of several key residues in the binding pocket. Rearrangements of side chains within the binding pocket accommodate the unique structural features of each siderophore. The backbones of the siderophores are not involved in any direct interactions with the protein, demonstrating how siderophores with considerable chemical and structural diversity can be bound by FhuD. For albomycin, which consists of an antibiotic group attached to a hydroxamate siderophore, electron density for the antibiotic portion was not observed. Therefore, this study provides a basis for the rational design of novel bacteriostatic agents, in the form of siderophore-antibiotic conjugates that can act as "Trojan horses," using the hydroxamate-type siderophore uptake system to actively deliver antibiotics directly into targeted pathogens.     

Characterisation of estrogenic 17beta-hydroxysteroid dehydrogenase (17beta-HSD) activity in the human brain

Estrogens play a crucial role in multiple functions of the brain and the proper balance of inactive estrone and active estradiol-17beta might be very important for their cerebral effects. The interconversion of estrone and estradiol-17beta in target tissues is known to be catalysed by a number of human 17beta-hydroxysteroid dehydrogenase (17beta-HSD) isoforms. The present study shows that enzyme catalysed interconversion of estrone and estradiol-17beta occurs in the human temporal lobe. The oxidative cerebral pathway preferred estradiol-17beta to Delta(5)-androstenediol and testosterone, whereas the reductive pathway preferred dehydroepiandrosterone (DHEA) to Delta(4)-androstenedione and estrone. An allosteric Hill kinetic for NAD-dependent oxidation of estradiol-17beta was observed, whereas a typical Michaelis-Menten kinetic was shown for NADPH-dependent reduction of estrone. Investigations of the interconversion of estrogens in cerebral neocortex (CX) and subcortical white matter (SC) preparations of brain tissue from 12 women and 10 men revealed no sex-differences, but provide striking evidence for the presence of at least one oxidative membrane-associated 17beta-HSD and one cytosolic enzyme that catalyses both the reductive and the oxidative pathway. Membrane-associated oxidation of estradiol-17beta was shown to be significantly higher in CX than in SC (P<0.05), whereas the cytosolic enzyme activities were significantly higher in SC than in CX (P<0.0005). Finally, real-time RT-PCR analyses revealed that besides 17beta-HSD types 4 and 5 also the isozymes type 7, 8, 10 and 11 show substantial expression in the human temporal lobe. The characteristics of the isozymes lead us to the conclusion that cytosolic 17beta-HSD type 5 is the best candidate for the observed cytosolic enzyme activities, whereas the data gave no clear answer to the question, which enzyme is responsible for the membrane-associated oxidation of estradiol-17beta. In conclusion, the study strongly suggests that different cell types and different isozymes are involved in the cerebral interconversion of estrogens, which might play a pivotal role in maintaining the functions of the central nervous system.     

Gene therapy: prospects for glycolipid storage diseases

Lysosomal storage diseases comprise a group of about 40 disorders, which in most cases are due to the deficiency of a lysosomal enzyme. Since lysosomal enzymes are involved in the degradation of various compounds, the diseases can be further subdivided according to which pathway is affected. Thus, enzyme deficiencies in the degradation pathway of glycosaminoglycans cause mucopolysaccharidosis, and deficiencies affecting glycopeptides cause glycoproteinosis. In glycolipid storage diseases enzymes are deficient that are involved in the degradation of sphingolipids. Mouse models are available for most of these diseases, and some of these mouse models have been used to study the applicability of in vivo gene therapy. We review the rationale for gene therapy in lysosomal disorders and present data, in particular, about trials in an animal model of metachromatic leukodystrophy. The data of these trials are compared with those obtained with animal models of other lysosomal diseases.