A contig of non-chimaeric YACs containing the spinal muscular atrophy gene in 5q13

We have constructed a contig of non-chimaeric yeast artificialchromosomes (YACs) across the candidate region for childhoodautosomal recessive spinal muscular atrophy (SMA) In 5q13. Anovel microsatellite reduces the candidate region to approximately400kb of DNA distal to D5S435. The candidate region containsblocks of chromosome 5 specific repeats which have copies on5p as well as elsewhere on 5q. Restriction mapping of the YACsreveals at least one CpG island In the SMA gene region. TheYAC maps indicate that the contig contains minimal rearrangementsor deletions. The data show the value of screening several YAClibraries simultaneously in order to construct a set of overlappingsequences suitable for candidate gene searches and direct genomicsequencing.     

Bioassay for growth hormone releasing hormone (GHRH) using a recombinant receptor and cAMP-responsive reporter system

Growth hormone releasing hormone (GHRH) receptors are members of the G-protein receptor family that use cAMP as a second messenger. A human fetal kidney 293-derived cell line stably expressing the porcine GHRH receptor (pGHRHr/293 cells) and a cAMP-responsive reporter system were used to develop a bioassay for human GHRH. The reporter system (ph180SEAP) was constructed by subcloning the tandem cAMP response elements from the human glycoprotein hormone subunit gene promoter (h180) upstream from the secreted alkaline phosphatase cDNA of reporter plasmid pSEAP-Basic. To generate a stable cell line expressing both the GHRH receptor and SEAP reporter system, a DNA fragment from pPUR that confers puromycin resistance was subcloned downstream from the reporter construct of ph180SEAP. Tranfection of ph180SEAPpur into pGHRHr/293 cells yielded pGHRHr/SEAP/293 cell lines that responded to recombinant GHRH with dose-dependent increases in SEAP activity. The GHRH receptor-SEAP reporter bioassay was compared to a conventional bioassay using cultured rat anterior pituitary cells. Synthetic and recombinant GHRH induced a 3.1-fold increase in growth hormone release by rat pituitary cells with ED₅₀'s of 3.6 and 2.2×10⁻¹⁰ M, respectively. Recombinant GHRH was 1.7±0.7 times more potent than synthetic GHRH in the pituitary cell bioassay. In an analogous experiment, pGHRHr/SEAP/293 cells responded to synthetic and recombinant GHRH with a 9.1-fold increase in SEAP activity. The ED₅₀'s were 7.8 and 4.3×10⁻¹¹ M, respectively, with recombinant GHRH being 1.8±0.1 times more potent than the synthetic preparation. Thus, the GHRH receptor-SEAP reporter bioassay is a sensitive, accurate, precise and efficient method for measuring GHRH biological activity.     

Modulatory role of alizarin from Rubia cordifolia L. against genotoxicity of mutagens

Rubia cordifolia L. (Rubiaceae) is an important medicinal plant used in the Ayurvedic medicinal system. Its use as a traditional therapeutic has been related to the treatment of skin disorders and cancer. Besides its medicinal value, anthraquinones from this plant are used as natural food colourants and as natural hair dyes. Dyes derived from natural sources have emerged as important alternatives to synthetic dyes. Alizarin (1,2-dihydroxyanthraquinone) was isolated and characterized from R. cordifolia L. and evaluated for its antigenotoxic potential against a battery of mutagens viz. 4-nitro-o-phenylenediamine (NPD) and 2-aminofluorene (2-AF) in Ames assay using TA98 tester strain of Salmonella typhimurium; hydrogen peroxide (H₂O₂) and 4-nitroquinoline-1-oxide (4NQO) in SOS chromotest using PQ37 strain of Escherichia coli and in Comet assay using human blood lymphocytes. Our results showed that alizarin possessed significant modulatory role against the genotoxicity of mutagens.     

Precursor arrays for triplet repeat expansion at the fragile X locus

To determine factors governing triplet repeat expansion at FMR1,we need to understand the basis of normal variation. We havesequenced the FMR1 repeat from 102 normal X chromosomes andshow that most are interrupted with a regularly spaced AGG trinucleotidegiving an ordered structure to the array. Five types of arrayswere identified consisting of varying numbers of a core unitwith consensus [AGG(CGG)_g]. Additional variation in the lengthof the (CGG)_n portion within each unit generates the continuumof lengths seen on normal chromosomes. Ten per cent containlong, uninterrupted tracts of (CGG)_n, and their lengths suggestthey have arisen by the loss of AGG triplets from longer interruptedarrays. Haplotype analysis of arrays carrying long, uninterrupted(CGG)_n tracts suggests that they occur more frequently on geneticbackgrounds which are more highly represented on fragile X chromosomes.These arrays may well be precursors from which the larger fragileX associated arrays have arisen by further expansion.     

Coordinated roles of ST3Gal-VI and ST3Gal-IV sialyltransferases in the synthesis of selectin ligands

Binding of selectins to their glycan ligands is a prerequisite for successful leukocyte trafficking. During synthesis and transport through the secretory pathway, selectin ligands are constructed with the participation of one or more sialyltransferases of the ST3Gal subfamily. Previous studies established that ST3Gal-IV only partially contributes to selectin ligand formation, indicating that other ST3Gal-sialyltransferases are involved. By generating and analyzing St3gal6-null mice and St3gal4/St3gal6 double-deficient mice, in the present study, we found that binding of E- and P-selectin to neutrophils and L-selectin binding to lymph node high endothelial venules is reduced in the absence of ST3Gal-VI and to a greater extent in double-deficient mice. In an ex vivo flow chamber assay, P- and E-selectin-dependent leukocyte rolling was mildly reduced in St3gal6-null mice and more severely in double-deficient mice. In inflamed cremaster muscle venules of St3gal6-null mice, we found impaired P-selectin-dependent, but not E-selectin-dependent leukocyte rolling, whereas in double-deficient mice, E-selectin-dependent rolling was almost completely absent. Furthermore, neutrophil recruitment into the inflamed peritoneal cavity and lymphocyte homing to secondary lymphoid organs were impaired in St3gal6-null mice and more severely in double-deficient mice. The results of the present study demonstrate the coordinated participation of both ST3Gal-VI and ST3Gal-IV in the synthesis of functional selectin ligands.     

Evaluation of antigenotoxic activity of isoliquiritin apioside from Glycyrrhiza glabra L.

Prevention of manifestation of events characteristic of carcinogenesis is being emphasized a rational strategy to combat cancer. Reactive oxygen species (ROS) play an important role in tumor initiation through oxidative damage of DNA. In search for lead molecules in cancer chemoprevention from natural products, a fraction ‘Rlicca’ isolated from Glycyrrhiza glabra was studied for modulatory effect against hydrogen peroxide and 4-nitroquinoline-N-oxide induced genotoxicity in Escherichia coli PQ37 using SOS chromotest and in human peripheral blood lymphocytes using the Comet assay. The fraction ‘Rlicca’ at a concentration of 191 μM decreased the SOS inducing potency (SOSIP) of hydrogen peroxide (1.0 mM) and NQO (20 μg/ml) by 83.72% and 68.77%, respectively. In the human blood lymphocytes, ‘Rlicca’ reduced the tail moment induced by hydrogen peroxide (25 μM) and NQO (5 μg/ml) by 88.04% and 76.64%, respectively, using the Comet assay. The spectroscopic data of ‘Rlicca’ fraction revealed it to be isoliquiritin apioside, a chalcone oligoglycoside. This is the first report of isoliquiritin apioside with marked potential to combat oxidative stress-induced genotoxicity.     

A rapid PCR method for genotyping the Large(myd) mouse, a model of glycosylation-deficient congenital muscular dystrophy

The myodystrophy (Large(myd)) mouse has a spontaneous loss of function mutation in a putative glycosyltransferase gene (Large). Mutations in the human gene (LARGE) have been described in congenital muscular dystrophy type 1D (MDC1D). Mutations in four other genes that encode known or putative glycosylation enzymes (POMT1, POMGnT1, fukutin and FKRP) are also associated with muscular dystrophy. In all these diseases hypoglycosylation of alpha-dystroglycan, and consequent loss of ligand binding, is a common pathomechanism. Currently, the Large(myd) mouse is the principal animal model for studying the underlying molecular mechanisms of this group of disorders. Over-expression of LARGE in cells from patients with mutations in POMT1 or POMGnT1 results in hyperglycosylation of alpha-dystroglycan and restoration of laminin binding. Thus, LARGE is a potential therapeutic target. Here, we define the intronic deletion breakpoints of the Large(myd) mutation and describe a simple, PCR-based diagnostic assay, facilitating the study of this important animal model.     

Inducing host protection in pneumococcal sepsis by preactivation of the Ashwell-Morell receptor

The endocytic Ashwell-Morell receptor (AMR) of hepatocytes detects pathogen remodeling of host glycoproteins by neuraminidase in the bloodstream and mitigates the lethal coagulopathy of sepsis. We have investigated the mechanism of host protection by the AMR during the onset of sepsis and in response to the desialylation of blood glycoproteins by the NanA neuraminidase of Streptococcus pneumoniae. We find that the AMR selects among potential glycoprotein ligands unmasked by microbial neuraminidase activity in pneumococcal sepsis to eliminate from blood circulation host factors that contribute to coagulation and thrombosis. This protection is attributable in large part to the rapid induction of a moderate thrombocytopenia by the AMR. We further show that neuraminidase activity in the blood can be manipulated to induce the clearance of AMR ligands including platelets, thereby preactivating a protective response in pneumococcal sepsis that moderates the severity of disseminated intravascular coagulation and enables host survival.Pathogens in the host bloodstream often induce a hyperactive coagulation cascade that can progress to disseminated intravascular coagulation with severe thrombosis, organ failure, and death (1–3). With current limited understanding of pathogen–host interactions, sepsis remains a debilitating and deadly syndrome with few treatment options (4, 5). An unexpected protective host response that reduces coagulopathy during sepsis caused by Streptococcus pneumoniae (SPN) was recently discovered in studies of the endocytic Ashwell-Morell receptor (AMR) (6, 7). Host protection by the AMR is linked to the hydrolysis of sialic acids from blood glycoproteins by the neuraminidase A (NanA) of SPN. Sialic acids are posttranslational glycan modifications often attached to underlying galactose on many cell surface and secreted glycoproteins (8, 9).Neuraminidases (aka sialidases) produced by microbial pathogens hydrolyze sialic acids on glycoproteins to establish infection and facilitate host colonization (10). For example, NanA remodels mucosal cell surface glycoproteins to promote bacterial colonization of the upper respiratory tract (11, 12) and contributes to pulmonary inflammation along with the development of SPN pneumonia (13, 14). However, the host has adapted to counteract the pathological effects of this SPN virulence factor by the clearance from blood circulation of host factors bearing AMR ligands that have been unmasked by NanA desialylation.In this study we have identified multiple blood components that are removed from circulation by the AMR and have determined which of these are primarily responsible for diminishing the lethal coagulopathy of SPN sepsis. We have further used this information to develop and assess a prophylactic approach that preactivates AMR function in the early phases of sepsis to reduce the severity and lethality of the ensuing coagulopathy.     

The cloning of FRAXF: trinucleotide repeat expansion and methylation at a third fragile site in distal Xqter

Three fragile sites, FRAXA, FRAXE and FRAXF lie in the Xq27–28region of the human X chromosome. The expression of FRAXA isassociated with the fragile X syndrome, the most prevalent formof Inherited mental retardation whilst the expression of FRAXEIs associated with a rarer and comparatively milder form ofmental handicap. Both the FRAXA and FRAXE sites have been clonedand the fragile site expression found to be due to the expansionof analogous CGG/GCC trinucleotide repeat arrays. We describehere the cloning of the third fragile site, FRAXF, and demonstratethat it Involves the expansion of a (GCCGTC)n(GCC)n compoundarray. PCR analyses across the repeat of normal individualsshow that the number of triplets in the array ranges from 12–26and the most common allele consists of 14 triplet units. Sequencinganalyses show that 95% of normal individuals have three copiesof the GCCGTC motif and In these individuals, the size variationobserved by PCR is due to copy number alterations in the GCCarray. In a cytogenetically positive male with developmentaldelay, the array is expanded by >900 triplets and the adjacentCpG-rich region is methylated. The array is also expanded Incytogenetically positive carrier females from the family originallyused to define the FRAXF site. We conclude that the expandedarray corresponds to the FRAXF fragile site.