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 byp>1p>H NMR spectroscopy and mass spectrometry. In the placental fraction substantial amounts of globotetraosylceramide (P-antigen) and globotriaosylceramide (Pp>kp>-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-Tjp>ap> serum is the placenta tissue, resulting in termination of the pregnancy.     

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, p786712405002m31/xxlarge947.gif" alt="gamma" align="MIDDLE" BORDER="0">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 p7r145k1878421t1/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-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 (p7r145k1878421t1/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-agonist and p7r145k1878421t1/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-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 p7r145k1878421t1/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-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 p7r145k1878421t1/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-receptor mediated process whereas AAT activation shows a mixed type of regulation where some well known p7r145k1878421t1/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-agonist and antagonists are behaving as p7r145k1878421t1/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-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.     

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.     

Levels of chromosomally encoded Umu proteins and requirements for in vivo UmuD cleavage

Summary Most of the inducible mutagenesis observed in Escherichia coli after treatment with many DNA damaging agents is dependent upon the products of the umuD,C operon. RecA-mediated proteolytic processing of UmuD yields a carboxyl-terminal fragment (UmuDp04u6134022/xxlarge8242.gif" alt="prime" align="BASELINE" BORDER="0">) that is active for mutagenesis. Processing of UmuD is therefore a critical step in the fixation of mutations. In this paper we have analyzed the requirements for UmuD processing in vivo. Standard immuno-detection assays, coupled with a sensitive chemiluminescence detection assay, have been utilized to probe levels of chromosomally encoded Umu proteins from whole-cell E. coli extracts. We found that the derepression of additional SOS gene products, other than RecA, was not required for UmuD processing. Moreover, efficient cleavage of UmuD was observed only in the presence of elevated levels of activated RecA, suggesting that efficient processing would occur only under conditions of severe DNA damage. Detection of chromosomally encoded Umu proteins has allowed us, for the first time, to measure directly the cellular steady-state levels of these proteins under various SOS inducing conditions. UmuD was present at p04u6134022/xxlarge8764.gif" alt="sim" align="MIDDLE" BORDER="0">180 copies per uninduced cell and was measured at p04u6134022/xxlarge8764.gif" alt="sim" align="MIDDLE" BORDER="0">2400 copies per cell in strains that lacked a functional repressor. Induced levels of UmuC were approximately 12-fold lower than UmuD with p04u6134022/xxlarge8764.gif" alt="sim" align="MIDDLE" BORDER="0">200 molecules per cell. These levels of cellular UmuC protein suggest that it functions through specific protein-DNA or protein-protein interactions, possibly as a lesion recognition protein or by interacting with DNA polymerase III.     

Carbon and nitrogen isotope ratios in different compartments of a healthy and a declining Picea abies forest in the Fichtelgebirge,NE Bavaria

Summary Natural carbon and nitrogen isotope ratios were measured in different compartments (needles and twigs of different ages and crown positions, litter, understorey vegetation, roots and soils of different horizons) on 5 plots of a healthy and on 8 plots of a declining Norway spruce (Picea abies (L.) Karst.) forest in the Fichtelgebirge (NE Bavaria, Germany), which has recently been described in detail (Oren et al. 1988a; Schulze et al. 1989). The p6652065/xxlarge948.gif" alt="delta" align="BASELINE" BORDER="0">p>13p>C values of needles did not differ between sites or change consistently with needle age, but did decrease from the sun-to the shade-crown. This result confirms earlier conclusions from gas exchange measurements that gaseous air pollutants did no long-lasting damage in an area where such damage was expected. Twigs (p6652065/xxlarge948.gif" alt="delta" align="BASELINE" BORDER="0">p>13p>C between-25.3 and-27.8p6652065/xxlarge8240.gif" alt="permil" align="MIDDLE" BORDER="0">) were significantly less depleted in p>13p>C than needles (p6652065/xxlarge948.gif" alt="delta" align="BASELINE" BORDER="0">p>13p>C between-27.3 and-29.1p6652065/xxlarge8240.gif" alt="permil" align="MIDDLE" BORDER="0">), and p6652065/xxlarge948.gif" alt="delta" align="BASELINE" BORDER="0">p>13p>C in twigs increased consistently with age. The p6652065/xxlarge948.gif" alt="delta" align="BASELINE" BORDER="0">p>15p>N values of needles ranged between-2.5 and-4.1p6652065/xxlarge8240.gif" alt="permil" align="MIDDLE" BORDER="0"> and varied according to stand and age. In young needles p6652065/xxlarge948.gif" alt="delta" align="BASELINE" BORDER="0">p>15p>N decreased with needle age, but remained constant or increased in needles that were 2 or 3 years old. Needles from the healthy site were more depleted in p>15p>N than those from the declining site. The difference between sites was greater in old needles than in young ones. This differentiation presumably reflects an earlier onset of nitrogen reallocation in needles of the declining stand. p6652065/xxlarge948.gif" alt="delta" align="BASELINE" BORDER="0">p>15p>N values in twigs were more negative than in needles (-3.5 to-5.2p6652065/xxlarge8240.gif" alt="permil" align="MIDDLE" BORDER="0">) and showed age- and stand-dependent trends that were similar to the needles. p6652065/xxlarge948.gif" alt="delta" align="BASELINE" BORDER="0">p>15p>N values of roots and soil samples increased at both stands with soil depth from-3.5 in the organic layer to +4p6652065/xxlarge8240.gif" alt="permil" align="MIDDLE" BORDER="0"> in the mineral soil. The p6652065/xxlarge948.gif" alt="delta" align="BASELINE" BORDER="0">p>15p>N values of roots from the mineral soil were different from those of twigs and needles. Roots from the shallower organic layer had values similar to twigs and needles. Thus, the bulk of the assimilated nitrogen was presumably taken up by the roots from the organic layer. The problem of separation of ammonium or nitrate use by roots from different soil horizons is discussed.     

Immunocytochemical localization of conglutin γ and legumin-like globulin in developing and mature seeds ofLupinus albus L.

Summary The distribution of two seed proteins, namely conglutin p178/xxlarge947.gif" alt="gamma" align="MIDDLE" BORDER="0"> and a legumin-like globulin, in developing and mature seeds ofLupinus albus L. has been examined by immunocytochemistry and the concomitant modifications of their constituent polypeptides followed by SDS-PAGE. Both proteins were found within vacuolar protein bodies in various tissues of the cotyledons, although with some differences in the distribution patterns. The legumin-like protein was found to be deposited within the large storage parenchyma cells of the cotyledons in a manner similar to that reported for other storage proteins; little or no immunolabelling was associated with the cotyledonary epidermal and vascular parenchyma cells. In contrast conglutin p178/xxlarge947.gif" alt="gamma" align="MIDDLE" BORDER="0"> was present in all cell types.A precursor of the legumin-like protein accumulated transiently in the developing cotyledon, but was subsequently modified by proteolytic cleavage. The onset of such modification was concomitant with a transition in the predominant vacuolar forms within the storage parenchyma cells. No precursor molecules of conglutin p178/xxlarge947.gif" alt="gamma" align="MIDDLE" BORDER="0"> have been detected in this study, thus indicating that this protein is deposited in the protein bodies in its mature form.Abbreviations LM light microscopy - EM electron microscopy - DAF days after flowering - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - GAR goat antirabbit antiserum     

Barley aleurone layer cell protoplasts as a transient expression system

Protoplasts were prepared from barley aleurone layers using p2w511g05756l72h/xxlarge8216.gif" alt="lsquo" align="BASELINE" BORDER="0">Onozukap2w511g05756l72h/xxlarge8217.gif" alt="rsquo" align="BASELINE" BORDER="0"> cellulase digestion and purification through a Percoll gradient. Protoplasts prepared by this procedure had a viability ranging from 60% to 80% during the first two days of culture. They were responsive to gibberellic acid (GA) as measured by the stimulation of p2w511g05756l72h/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-amylase synthesis. The GA stimulation was counteracted by abscisic acid (ABA). In the presence of polyethylene glycol (PEG), the protoplasts took up exogenously added plasmid DNA containing the reporter gene coding for chloramphenicol acetyl transferase (CAT) linked to a 35S promoter from cauliflower mosaic virus (CaMV) or to barley p2w511g05756l72h/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-amylase gene promoters and expressed CAT activity. Therefore, barley aleurone layer protoplasts are suitable for analysis of hormoneresponsive elements in hydrolase genes.     

A detailed study of the regulation and evolution of the two classes of patatin genes in Solanum tuberosum L.

The class-specific expression of patatin genes was investigated by analysing four new patatin genes. A class I patatin gene from cv. Berolina as well as a class I and two class II patatin genes from the monohaploid cultivar AM 80/5793 were isolated and partially sequenced. Sequence comparison indicates rearrangements as the major source for the generation of diversity between the different members of the classes. The expression of single genes was studied in potato plants transformed with chimaeric genes where the putative patatin promoters were fused to the GUS reporter gene. A detailed histochemical analysis reveals that both class I genes are expressed as the previously described class I patatin gene B33 from cv. Berolina [1], i.e. in the starch-containing cells of potato tubers and in sucrose-induced leaves. The class II gene pgT12 shows the same pattern as the previously described class II gene pgT2 [2], i.e. expression in root tips and in the vascular tissue of tubers, whereas no activity was detectable for pgT4. Thus the expression pattern of both classes of genes seems to be stable at least within or even between different cultivars.     

An optimized protocol for the production of high purity maltose

An economical protocol, which is simple, rapid and reproducible for the production of maltose by enzymatic hydrolysis of tapioca starch, has been optimized. The protocol involves liquefaction of 35% (w/w) tapioca starch by bacterial p526368u071/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-amylase at 78±2°C to 3 to 5% (w/w) reducing sugars, followed by maximal (85±3% w/w maltose equivalent) saccharification with barley p526368u071/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-amylase and pullulanase at 50°C for 24 to 30 h. The post-saccharification recovery protocol comprised decolourization by charcoal, de-dextrinization by denatured spirit precipitation, de-ionization by passage through cation and anion exchangers and dehydration by vacuum drying. A white crystalline maltose powder was obtained with specifications comparable to commercial high purity maltose. The protocol yields at least 60% (w/w) recovery of maltose and is suitable for use by the pharmaceutical industry. The protocol is unique in that it utilizes cheap and easily hydrolysed tapioca starch, leaves no mother liquor, enabling higher recovery of maltose, and allows almost quantitative recovery of limit maltodextrins, a value-added marketable by-product.