Imprinting and X chromosome counting mechanisms determine Xist expression in early mouse development.

In mice, X inactivation is preceded by in cis Xist expression. Initially, normal female embryos express the paternal Xist allele exclusively, preceding imprinted X inactivation in the trophectoderm. Later expression of Xist alleles is random, preceding random X inactivation in the epiblast lineage.

Evidence that random and imprinted Xist expression is controlled by preemptive methylation.

The mouse Xist gene is expressed exclusively from the inactive X chromosome and may control the initiation of X inactivation. We show that in somatic tissues the 5' end of the silent Xist allele on the active X chromosome is fully methylated, while the expressed allele on the inactive X is completely unmethylated. In tissues that undergo imprinted paternal Xist expression and imprinted X inactivation, the paternal Xist allele is unmethylated, and the silent maternal allele is fully methylated.

Sulfated blood group Lewis(a). A superior oligosaccharide ligand for human E-selectin.

In earlier studies of oligosaccharide probes (neoglycolipids) generated from an ovarian cystadenoma glycoprotein, one of the components that strongly supported binding of the endothelial adhesion molecule, E-selectin, was identified as an equimolar mixture of tetrasaccharides of blood group Le(a) and Le(x) type sulfated at position 3 of the outer galactose (C.-T. Yuen, A.

Enzymological and mutational analysis of a complex primary hyperoxaluria type 1 phenotype involving alanine:glyoxylate aminotransferase peroxisome-to-mitochondrion mistargeting and intraperoxisomal aggregation.

Primary hyperoxaluria type 1 (PH1) is a rare autosomal recessive disease caused by a deficiency of the liver-specific peroxisomal enzyme alanine:glyoxylate aminotransferase (AGT). Three unrelated PH1 patients, who possess a novel complex phenotype, are described. At the enzymological level, this phenotype is characterized by a complete, or nearly complete, absence of AGT catalytic activity and reduced AGT immunoreactivity.

Acute effects of ethanol on hepatic endocytosis and processing of insulin in perfused rat liver.

This study utilizes the perfused rat liver combined with subcellular fractionation and compartmental analysis to investigate the effects of ethanol on hepatic uptake, endocytosis, and processing of insulin. At 4 min after the start of a 2-min pulse of radiolabeled insulin, increasing ethanol concentrations progressively inhibited insulin uptake by the liver (57% at 50 mM ethanol). Subcellular fractionation of the perfused livers showed a progressive shift in distribution from a predominantly endosomal location (control) to a bimodal distribution between endosomes and plasma membrane.

Expression of Xist during mouse development suggests a role in the initiation of X chromosome inactivation.

The mouse Xist gene maps to the X inactivation center (Xic) region and is expressed exclusively from the inactive X chromosome. It is thus a candidate gene for the Xic. We show that the onset of Xist expression in mouse development precedes X chromosome inactivation and may therefore be a cause rather than merely a consequence of X inactivation.

The product of the mouse Xist gene is a 15 kb inactive X-specific transcript containing no conserved ORF and located in the nucleus.

The Xist gene maps to the X inactivation center region in both mouse and human, and previous analysis of the 3' end of the gene has demonstrated inactive X-specific expression, suggesting a possible role in X inactivation. We have now analyzed the entire mouse Xist gene. The mature inactive X-specific transcript is 15 kb in length and contains no conserved ORF.

Thermogenesis after surgery: effect of perioperative heat conservation and epidural anesthesia.

Body temperature, respiratory gas exchange, and plasma catecholamines were determined before and after surgery in three groups [control (C), warmed (W), and epidural (E) who received local anesthetic at T4-S5 dermatomes during and for 24 h after surgery] of patients undergoing colonic surgery under general anesthesia. At the end of surgery, group W were nursed in an ambient temperature of 28-30 degrees C, whereas the others were at 20-23 degrees C for a period of 24 h. Core (Tc) and dorsal hand temperature decreased during surgery in both C and E (P less than 0.

Characterisation by mass spectrometry and 1H-NMR of novel hexasaccharides among the acidic O-linked carbohydrate chains of bovine submaxillary mucin.

The acidic oligosaccharide alditols released from bovine submaxillary-gland mucin by Carlson degradation were investigated by a combination of liquid secondary-ion mass spectrometry, methylation analysis and 1H-NMR. Among the largest structures identified were four branched hexasaccharides, three of them novel, comprising two separate pairs of structures. One pair contained the sequence Fuc(alpha 1-2)Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc(beta 1-) (Fuc, L-fucose), at C3 of N-acetylgalactosaminitol and differed only by substitution at C6 by N-acetylneuraminic or N-glycolylneuraminic acid.

Spectrum of sialylated and nonsialylated fuco-oligosaccharides bound by the endothelial-leukocyte adhesion molecule E-selectin. Dependence of the carbohydrate binding activity on E-selectin density.

Carbohydrate recognition by the human endothelial-leukocyte adhesion molecule, E-selectin, has been investigated by binding studies using 3H-labeled Chinese hamster ovary cells expressing different levels of the transfected full-length adhesion molecule and a series of structurally defined oligosaccharides linked to the lipid phosphatidylethanolamine dipalmitoate (neoglycolipids) and synthetic glycolipids chromatographed on silica gel plates or immobilized on plastic wells. Evidence is presented for density-dependent binding of the membrane-associated E-selectin not only to 3'-sialyl-lacto-N-fucopentaose II (3'-S-LNFP-II) and 3'-sialyl-lacto-N-fucopentaose III (3'-S-LNFP-III) which express the sialyl Le(a) and sialyl Le(x) antigens, respectively, but also to the nonsialylated analogue LNFP-II; there is a threshold density of E-selectin required for binding to these sialylated sequences, and binding to the nonsialylated analogue is a property only of cells with the highest density of E-selectin expression. The presence of fucose linked to subterminal rather than to an internal N-acetylglucosamine is shown to be a requirement for E-selectin binding, and although the presence of sialic acid 3-linked to the terminal galactose of the LNFP-II or LNFP-III sequences substantially enhances E-selectin binding, the presence of 6-linked sialic acid abolishes binding.

Regulation of extracellular adenosine production by ectonucleotidases of adult rat ventricular myocytes.

We have investigated the kinetic properties of the extracellular reaction sequence ATP----ADP----AMP----adenosine catalyzed by ectonucleotidases at the surface of adult rat cardiac myocytes. Analysis of progress of reaction curves indicates that depletion of substrate at cell surfaces dominates the regulation of the rate of hydrolysis of ATP or of ADP when it is the initial substrate. Preferential delivery of intermediate products to be substrates at cell surfaces makes a significant contribution to the regulation of adenosine production from ATP or ADP.

Growth retardation and immunodeficiency in a patient with mutations in the DNA ligase I gene.

DNA ligases are required for the replication, repair, and recombination of DNA. A cell line derived from a young woman with growth retardation, sun sensitivity, and immunodeficiencies, who died aged 19 with lymphoma, showed two different miscoding mutations at the DNA ligase I locus on chromosome 19, one in each allele. One of the mutations was inherited from the mother and has also been detected in two healthy brothers.

Subunit molecular mass assignment of 14,654 Da to the soluble beta-galactoside-binding lectin from bovine heart muscle and demonstration of intramolecular disulfide bonding associated with oxidative inactivation.

The soluble dimeric beta-galactoside-binding lectin (subunit molecular mass, approximately 14 kDa) of bovine heart muscle, in common with the 14-kDa lectins of several other animal species, displays carbohydrate-binding activity when it is in the reduced state, but the purified lectin loses this activity upon oxidation. In the present study, the presence of any post-translational modification and the mechanism of the oxidative inactivation have been investigated by analyses of the reduced and oxidized forms of the purified bovine lectin by electrospray ionization-mass spectrometry (ESI-MS) and by liquid secondary ion mass spectrometry (LSIMS) of tryptic and peptic peptides. By ESI-MS, the molecular mass of the reduced lectin is determined to be 14,654.

Familial recurrence-pattern analysis of cleft lip with or without cleft palate.

Cleft lip with or without cleft palate (CL/P) is a common congenital malformation with an incidence in European white populations of about 1/1,000. The familial clustering of CL/P has been extensively characterized, and epidemiological studies have proposed monogenic models (with reduced penetrance), multifactorial/threshold models, and mixed major-gene/multifactorial models to explain its inheritance. The recognition of an association between two RFLPs at the transforming growth factor alpha (TGFA) locus and CL/P supports a major-gene component to the etiology of CL/P.

Sequence requirements for the editing of apolipoprotein B mRNA.

Apolipoprotein (apo) B48 is produced in the mammalian intestine by a tissue-specific RNA-editing mechanism, which mediates a C to U conversion at position 6666 in apoB mRNA. This generates an inframe translation stop codon (UAA) in place of glutamine (CAA) at position 2153. To establish the sequences required for editing we have used an in vitro conversion assay to monitor the editing of synthetic RNAs by rat intestinal extracts.

The effect of elemental diet on intestinal permeability and inflammation in Crohn's disease.

This study examines whether treatment of acute Crohn's disease with an elemental diet improves intestinal integrity and inflammation as assessed by a 51Cr-labeled ethylenediaminetetraacetatic acid (EDTA) permeability test and the fecal excretion of 111In-labeled autologous leukocytes, respectively. Thirty-four patients with active Crohn's disease completed a 4-week treatment course with an elemental diet. Active disease was characterized by increased intestinal permeability [24-hour urine excretion of orally administered 51Cr-EDTA, 6.

Potent vasoactive properties of endothelin 1 in human skin.

The effect of the endothelial cell-derived peptide endothelin 1 was investigated in human skin. Intradermal injection of endothelin 1 (1-100 pmol) caused a dose-dependent area of pallor that was associated with a significant reduction in basal skin blood flow, measured by laser-Doppler blood flowmeter (with 1 pmol endothelin, P = 0.012, analysis of variance).

Microscale sequencing of O-linked oligosaccharides using mild periodate oxidation of alditols, coupling to phospholipid and TLC-MS analysis of the resulting neoglycolipids.

In the course of characterising neoglycolipid products derived from mucin oligosaccharide alditols after periodate oxidation and coupling by reductive amination to the aminolipid dipalmitoylglycerophosphoethanolamine, we have obtained evidence that oxidative cleavage occurs specifically at the C4-C5 bond of core N-acetylgalactosaminitol. Two lipid-linked fragments thus obtained from each oligosaccharide alditol are well resolved on thin-layer chromatography and can be sensitively analysed by liquid-secondary-ion mass spectrometry to assign the sequence and branching patterns of oligosaccharides linked at C6 and C3 to the N-acetylgalactosamitol. These conclusions have been reached from detailed studies of the neoglycolipid derivatives of several oligosaccharides (di- to hexasaccharides) which were isolated from human meconium and characterised previously by MS and NMR studies as the free alditols.

Use of bacterial expression cloning to localize the epitopes for a series of monoclonal antibodies against apolipoprotein B100.

Bacterial expression of apolipoprotein (apo) B cDNA constructs has been used to map a series of monoclonal antibodies (mAbs) to apoB by immunoblotting. In some cases assignments have been confirmed and refined by (i) semipurification of expressed protein, CNBr digestion, and assignment of the immunoreactive fragments; (ii) controlled digestion of the cDNA with the exonuclease Bal31 and bacterial expression of the truncated proteins that result; or (iii) expression of specific segments of cDNA amplified by the polymerase chain reaction. Forty mAbs were mapped to a minimum of 17 separate determinants on apoB.

A library of oligosaccharide probes (neoglycolipids) from N-glycosylated proteins reveals that conglutinin binds to certain complex-type as well as high mannose-type oligosaccharide chains.

This report describes the preparation of a library of oligosaccharide probes (neoglycolipids) from N-glycosylated proteins, characterization of the probes by liquid secondary ion mass spectrometry, and investigation of their reactions with 125I-labeled bovine serum conglutinin by chromatogram binding assays. The results, together with additional binding studies using neoglycolipids derived from purified complex type bi-, tri-, and tetraantennary oligosaccharides from urine, or their glycosidase-treated products, have shown that the combining specificity of conglutinin includes structures not only on high mannose-type oligosaccharides but also on hybrid- and complex-type chains. With high mannose-type oligosaccharides there is increased reactivity from the Man5 to the Man8 structures, indicating a preference for the terminal Man alpha 1-2 sequence.

Kinetics of adenine nucleotide catabolism in coronary circulation of rats.

We have used the rat isolated, perfused heart to study the metabolism of adenine nucleotides on a single passage through the coronary circulation. Low doses (3-30 nmol) of ATP, ADP, or AMP injected as a bolus were extensively catabolized by ectoenzymes. Increasing doses of each nucleotide demonstrated saturability of catabolism that occurred at significantly lower doses of AMP than of ADP or ATP.

Hepatic processing of insulin. Characterization of differential inhibition by weak bases.

The effect of selected weak bases on the subcellular distribution and processing of internalized insulin by the liver has been studied. The effect of these bases on both the degradation products formed and on the kinetics of degradation have also been studied. 1.

Primary stimulation by dendritic cells induces antiviral proliferative and cytotoxic T cell responses in vitro.

We used well-gassed hanging drop (20 microliters) cultures with high concentrations of purified T cells from normal BALB/c mice to examine whether dendritic cells (DC) can induce primary antiviral proliferative T cell responses and generate virus-specific CTL. We found that DC exposed to infectious influenza virus in vitro or in vivo in small numbers (0.1-1%) resulted in strong proliferation of responder T cells within 3 d, and this was strongly inhibited by antibodies to class II MHC molecules.

Lysosomal hydrolases of human vascular cells: response to agonists of endothelial function.

Endothelial injury has been proposed as a feature of a wide variety of vascular diseases, and release of endothelial lysosomal hydrolases could contribute to the pathological changes seen. We have determined the relative activities of 14 glycosidases, two esterases and four peptide hydrolases in human umbilical vein endothelial cells and investigated whether known agonists of endothelial function, or materials known to modulate hydrolase secretion in other phagocytic cells, influenced the activity or secretion of these enzymes by human umbilical vein endothelial cells. Hexosaminidase, beta-galactosidase, beta-glucuronidase and alpha-iduronidase accounted for most of the measured glycosidase activity.