Glycosphingolipid expression in spontaneously aborted fetuses and placenta from blood group p women. Evidence for placenta being the primary target for anti-Tja-antibodies

A 12-week-old fetus and one 17-week-old fetus + placenta were obtained after spontaneous abortions from two women of blood group p. The 17-week-old fetus was dissected into intestine, liver, brain and residual tissue. Nonacid glycosphingolipid fractions were prepared from the tissues. Glycolipid characterization was carried out using thin layer chromatography immunostained with monoclonal antibodies and bacteria and by¹H NMR spectroscopy and mass spectrometry. In the placental fraction substantial amounts of globotetraosylceramide (P-antigen) and globotriaosylceramide (P^k-antigen) were identified. In contrast, the fetuses contained only trace amounts of these structures, as revealed by immunostaining. These results indicate that the primary target for the antibodies of the anti-Tj^a serum is the placenta tissue, resulting in termination of the pregnancy.     

Purification of the Lewis blood-group gene associated α-3/4-fucosyltransferase from human milk: an enzyme transferring fucose primarily to Type 1 and lactose-based oligosaccharide chains

A soluble Lewis blood-group gene associated -3/4-L-fucosyltransferase has been purified from human milk by a series of steps involving hydrophobic chromatography on Phenyl Sepharose 4B, ion exchange chromatography on CM-Sephadex C-50, affinity chromatography on GDP-hexanolamine Sepharose 4B and gel filtration on Sephacryl S-200. The first step separated -3-L-fucosyltransferase activity directed towardsN-acetylglucosamine in Type 2 (Gal1-4GlcNAc-R) acceptors from an -3/4-fucosyltransferase fraction acting on both Type 1 (Gal1-3GlcNAc-R) and Type 2 acceptors. Further purification of this latter fraction on CM-Sephadex and GDP-hexanolamine Sepharose gave a single peak of fucosyltransferase activity that catalysed the addition of fucose toN-acetylglucosamine in both Type 1 and Type 2 acceptors and to theO-3 position of glucose in lactose-based oligosaccharides. The enzyme preparation at this stage resembled previously described -3/4-fucosyltransferase preparations purified from human milk. However, gel filtration of this preparation on Sephacryl S-200 or Sephadex G-150 separated further amounts of -3-fucosyltransferase activity acting solely on Type 2 acceptors and left a residual -3/4-fucosyltransferase that retained strong -4 activity with the Type 1 acceptor, lacto-N-biose 1, and -3 activity with 2-fucosyllactose, but had relatively little -3 activity withN-acetyllactosamine and virtually no capacity to transfer fucose to glycoproteins withN-linked oligosaccharide chains having unsubstituted terminal Type 2 structures.     

Expression of c-myc protooncogene in rat lens cells during development,maturation and reversal of galactose cataracts

It is well established that normal patterns of epithelial cell proliferation and metabolism, and of fiber cell differentiation and maturation are essential for the maintenance of transparency in the ocular lens. Several factors, including exposure to high levels of sugars, have been known to result in the compromise of lens transparency. For example, initiation of lens cell damage by galactose induces lens epithelial cells to proliferate. Elevated levels of c-myc mRNA have usually been correlated with rapid cell growth and increased entry of cells into the S phase. Therefore, changes in c-myc mRNA levels may provide an early indication of the stimulation of lens epithelial cells to proliferate and differentiate, which has been postulated to be an early and important event in response to lens cell injury by galactose. By Northern blot hybridization analysis we quantitated c-myc mRNA levels in the lens capsule epithelia of rats (1) exposed to galactose, and (2) undergoing a partial recovery from the galactose-induced cell damage. At the onset of lens cell damage, we find c-myc mRNA to elevate to 6-fold by 24 hr, and by 48 hr decreases to about 3-fold the normal levels. During recovery, c-myc mRNA continues to be expressed at high levels approaching a 10-fold increase by day 12, then decreasing to levels of about 8-fold the control by day 30. The 24 h transitory elevation in c-myc mRNA in lens epithelial cells is in accord with our previous observations on the 24 h increase in MP26, crystallin and aldose reductase mRNAs following a high influx of galactose. Therefore, the elevation in c-myc mRNA as well suggest that galactose appears to cause lens cells to undergo an early transitory period of gene induction following the exposure of lens cells to galactose.     

Effect of adrenergic agonists and antagonists on alanine amino transferase,fructose-1:6-bisphosphatase and glucose production in hepatocytes

Using rat hepatocytes we confirmed our previous results that glucagon and -adrenergic agonists increased the enzyme activity of alanine aminotransferase (AAT) and propranolol abolished their effects. Only the enzyme activity was measured and other parameters like quantity of the enzyme or activation due to modification were not looked for. As in perfusion experiment phenylephrine and phenoxybenzamine (-agonist and -antagonist respectively) also increased the AAT activity in isolated rat hepatocytes and propranolol reversed these effects. The additive effect of glucagon and phenoxybenzamine on AAT was also persistant in hepatocyte system.Fructose- 1:6-bisphosphatase (Fru-P₂ase), another key enzyme in gluconeogenic pathway, was elevated by glucagon and other -adrenergic agonists both in liver perfusion and isolated hepatocyte experiments and was brought back to the normal level by propranolol. In this case also only the enzyme activity was measured and no other parameters were looked for. Unlike AAT this enzyme was not stimulated by phenylephrine or phenoxybenzamine. But AAT and Fru-P₂-ase activities were increased significantly by adenylate cyclase activators like fluoride or forskolin. Thus, it appears that the regulation of fru-P₂-ase by glucagon is purely a -receptor mediated process whereas AAT activation shows a mixed type of regulation where some well known -agonist and antagonists are behaving as -agonists.Results further indicate the presence of phosphodiesterase in hepatocyte membrane which was stimulated by glucagon and brought back to the normal level by propranolol.The different adrenergic compounds stated above, not only modified the activity of the above two enzymes but also stimulated glucose production by hepatocytes from alanine which was in turn abolished by propranolol as well as amino oxyacetate (AOA), a highly specified inhibitor of AAT. This confirm the participation of AAT in gluconeogenesis from alanine in liver. Forskolin and fluoride also increased the glucose production from alanine and showed additive effects with glucagon, phenylephrine and phenoxybenzamine.     

Alpha-interferon in Kikuchi’s disease

In Japan, histiocytic necrotizing lymphadenitis (Kikuchi’s disease) is a relatively common reactive lesion affecting lymph nodes, but the histogenesis and pathogenesis of the disease have not been clarified. Alpha-interferon has a role in the body’s defense against, viral infections. Using a polyclonal antibody to human alpha-interferon, we found numerous cells, mainly histiocytes, containing alpha-interferon in affected foci in the lymph nodes from 24 patients with Kikuchi’s disease. Tubuloreticular structures, thought by some authors to be associated with the production of interferon, were detected by electron microscopy in histiocytes, activated lymphocytes and vascular endothelial cells in the affected foci. These results suggested that the formation of tubuloreticular structures is a secondary phenomenon following stimulation by alpha-interferon. Further, the activity of 2′–5′ oligoadenylate synthetase, which is induced by alpha-interferon and enhanced during the early or active stage of viral infection, showed increased levels of activity in the active stage of Kikuchi’s disease and decreased to normal levels in the convalescent stage 2 weeks later. These results suggested the possibility of a viral etiology for Kikuchi’s disease.     

The distribution of A1 adenosine receptor and 5′-nucleotidase in pig brain cortex subcellular fractions

Pig brain cerebral cortex was subfractionated by isopycnic centrifugation in sucrose gradients. In each subfraction the content of the agonist [³H]R-PIA binding, the activity of adenosine metabolizing enzymes (5-nucleotidase and adenosine deaminase) and the activity of membrane marker enzymes were determined. The fractions were also examined by electron microscope. In general, the results suggest a widespread distribution of A₁ adenosine receptors in membranes from different origins. Marker enzyme profile characterization indicated an enrichment of A₁ adenosine receptor in pre-synaptic membranes isolated from the crude synaptosomal fraction (P2B subfraction) as well as in membranes of glial origin such as myelin. The receptor is also present in the endoplasmic reticulum and in membranes isolated from the microsomal fraction that seem to have a post-synaptic origin (P3B). In subfractions having a high content of adenosine receptor the equilibrium binding paramters were obtained as well as the proportion of high- to low-affinity sites. From the values of the equilibrium constants it was not possible to find differences between the receptor in the different subfractions. Analysis of the affinity state distribution showed a diminished percentage of high-affinity sites in fraction P3A, which can be accounted by the existence of myelin membranes; in contrast the percentage of high-affinity states was higher in P2 and P3B, indicating that in these fractions the receptor is present in synaptosomal membranes. The close correlation shown between the enzyme 5-nucleotidase specific activity and the specific ligand binding distributions led us to postulate an important role for the enzyme in the regulation of adenosine action in pig brain cortex.     

NMDA受体在海马CA_3区习得性LTP保持中的作用

在大鼠明暗分辨学习的建立和巩固过程中,通过与记录电极一起慢性埋植于海马CA_3区的注药管微量注射NMDA受体的特异性拮抗剂2-amino-5-phosphonovaleric acid(APV),观察对海马CA_3区突触效应及与之相关的习得性行为的影响。结果如下:(1)在动物经训练PS峰幅值刚增大至最高水平后,即在习得性LTP刚好形成后,每实验单元先于CA_3区注射AFV 1μl(2mmol/L),然后在药物有效作用时间内再进行训练,则PS峰值不能随训练而保持在最高水平,相反经8个实验单元,PS峰值降至实验前水平;相应地动物的正确反应率不能随训练而巩固,反而下降至10％以下。(2)在动物习得性LTP已形成并经一单元训练PS保持在最高水平后,于每实验单元训练前注射APV 1μl(2mmol/L),PS峰值同样不会随训练而保持在最高水平,经14个实验单元注药和训练,PS峰值逐渐降至实验前水平,相应地动物行为的正确反应率也降至10％以下,习得性行为消退,不过其消退速度比前一情况的动物为慢,说明习得性LTP发展情况不同,APV的作用效率有差别。结果表明:NMDA受体在习得性LTP的巩固中起着重要作用。     

The 5S DNA units of bread wheat (Triticum aestivum)

A collection of 44 cloned 5S DNA units fromTriticum aestivum cv. Chinese Spring were grouped into 12 sequence-types based on sequence similarity and the respective consensus sequences were then produced. The relationship between these 12 consensus sequences (T. aestivum S 1-S 8 andT. aestivum L 1-L 4), together with two clones sequenced byGerlach andDyer, and the 5S DNA consensus sequences from diploidTriticum spp. were then determined by numerical methods. Both phenetic and cladistic analyses were carried out. The following wheat 5S DNA sequences were found to group with respective sequences from diploidTriticum spp.:T. aestivum S 4, S 6 withT. tauschii S;T. aestivum S 3 withT. monococcum S andT. monococcum S-Rus 7;T. aestivum L 1 andT. aestivum L-G&D withT. speltoides L;T. aestivum L 2, L 3 withT. tauschii L;T. aestivum L 4 withT. monococcum L andT. monococcum L-Rus 12. The analyses suggested that 5 out of the 65S Dna loci present in wheat were identified at the sequence level. The locus that could not be identified in this analysis was the5S Dna-B 1 locus. A group ofT. aestivum sequences (T. aestivum S 1, S 7, S 8, S-G&D) were found to be distinct from the other 5S DNA sequences in the data base. The existence of the distinct group of 5S DNA sequences suggests that there is a gap in our current understanding of wheat evolution with respect to the5S Dna loci.     

Relationships betweenOryza species (Poaceae) based on 5S DNA sequences

Relationships between 9Oryza species, covering 6 different genomes, have been studied using hybridization and nucleotide sequence information from the5S Dna locus. Four to five units of the major size class of 5S DNA in each species, 55 units in all, were cloned and sequenced. Both hybridization and sequence data confirmed the basic differences between the A and B, C, D genome species suggested by morphological and cytological data. The 5S DNA units of the A genome species were very similar, as were the ones from the B, C, and D genome-containing species. The 5S DNA ofO. australiensis (E genome) grouped with the B, C, D cluster, while the units ofO. brachyantha (F genome) were quite different and grouped away from all other species. 5S DNA units fromO. minuta, O. latifolia, O. australiensis, andO. brachyantha hybridized strongly, and preferentially, to the genomic DNA from which the units were isolated and hence could be useful as species/genome specific probes. The 5S DNA units fromO. sativa, O. nivara, andO. rufipogon provided A genome-specific probes as they hybridized preferentially to A genome DNA. The units fromO. punctata andO. officinalis displayed weaker preferential hybridization toO. punctata DNA, possibly reflecting their shared genome (C genome).     

Comparison between avian and human prolyl 4-hydroxylases: studies on the holomeric enzymes and their constituent subunits.

Prolyl 4-hydroxylase, a key enzyme in collagen biosynthesis, catalyzes the conversion of selected prolyl residues to trans-hydroxyproline in nascent or completed pro-alpha chains of procollagen. The enzyme is a tetramer composed of two nonidentical subunits, designated alpha and beta. To compare the enzyme and its subunits from different sources, the chick embryo and human placental prolyl 4-hydroxylases were purified to homogeneity and their physicochemical and immunological properties were determined. Both enzymes were glycoproteins with estimated apparent molecular weights ranging between 400 and 600 kDa. Amino acid and carbohydrate analyses showed slight differences between the two holomeric enzymes, consistent with their deduced amino acid sequences from their respective cDNAs. Human placental prolyl 4-hydroxylase contained more tightly bound iron than the chick embryo enzyme. Immunodiffusion of the human placental enzyme with antibodies raised against the purified chick embryo prolyl 4-hydroxylase demonstrated partial identity, indicating different antigenic determinants in their tertiary structures. The enzymes could be separated by high-resolution capillary electrophoresis, indicating differential charge densities for the native chick embryo and human placental proteins. Electrophoretic studies revealed that the human prolyl 4-hydroxylase is a tetrameric enzyme containing two nonidentical subunits of about 64 and 62 kDa, in a ratio of approximately 1 to 2, designated alpha and beta, respectively. In contrast, the chick embryo alpha and beta subunit ratio was 1 to 1. Notably, the human alpha subunit was partially degraded when subjected to electrophoresis under denaturing conditions. Analogously, when the chick embryo enzyme was subjected to limited proteolysis, selective degradation of the alpha subunit was observed. Finally, only the alpha subunit was bound to Concanavalin A demonstrating that the alpha subunits of prolyl 4-hydroxylase in both species were glycosylated. Using biochemical techniques, these results demonstrated that the 4-trans-hydroxy-L-proline residues in human placental collagens are synthesized by an enzyme whose primary structure and immunological properties differ from those of the previously well-characterized chick embryo enzyme, consistent with their recently deduced primary structures from cDNA sequences.