Comparative study of the sugar chains of factor VIII purified from human plasma and from the culture media of recombinant baby hamster kidney cells.

The asparagine-linked sugar chains of blood coagulation factor VIII preparations purified from human plasma of blood group A donors and from the culture media of recombinant BHK cells were released as oligosaccharides by hydrazinolysis. These sugar chains were converted to radioactive oligosaccharides by reduction with sodium borotritide and separated into neutral and acidic fractions by paper electrophoresis. Most of the acidic oligosaccharides were converted to neutral ones by sialidase digestion, indicating that they are sialyl derivatives. The neutral and sialidase-treated acidic oligosaccharides were fractionated by serial chromatography on immobilized lectin columns and Bio-Gel P-4 column. Structural study of each oligosaccharide by sequential exo- and endoglycosidase digestion and by methylation analysis revealed that both factor VIII preparations contain mainly high mannose-type and bi-, tri-, and tetra-antennary complex-type sugar chains. Some of the biantennary complex-type sugar chains from human plasma factor VIII contain blood group A and/or H determinant, while those from recombinant product do not. Some of the bi-, tri- and tetra-antennary complex-type sugar chains of the recombinant factor VIII contain the Gal alpha 1----3Gal group. A small number of the triantennary complex-type sugar chains from both preparations was found to contain the Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----4 (Gal beta 1----4GlcNAc beta 1----2)Man group. Studies of pharmacokinetic parameters of the recombinant factor VIII infused into baboons revealed that its half-life in blood circulation is similar to that of plasma derived factor VIII, suggesting that the oligosaccharide structural differences between them do not affect the fate of factor VIII in vivo.     

Studies of the sites of intracellular degradation of apolipoprotein B in Hep G2 cells.

We previously reported that treatment of Hep G2 cells with oleate significantly increased apolipoprotein B (apoB) secretion by reducing early intracellular degradation of nascent apoB. In the current study, inhibitors of secretory protein transport (brefeldin A and monensin), cell fractionation studies, and protease protection assays were utilized to determine the location of apoB degradation and to better define the mechanism whereby oleate treatment reduces nascent apoB intracellular degradation. When cells were treated with brefeldin A, which blocks endoplasmic reticulum (ER) to Golgi protein transport, apoB degradation continued in control cells, suggesting that apoB is degraded in the ER. When oleate-treated cells were blocked with brefeldin A, oleate failed to protect apoB from intracellular degradation. The effects of brefeldin A were not due to effects on lipid synthesis as brefeldin A did not inhibit the synthesis of triglyceride, phospholipid, free cholesterol, or cholesteryl ester in control cells and did not prevent the increases in triglyceride (14-fold) and phospholipid (1.4-fold) synthesis seen in oleate-treated cells. Simultaneous treatment of cells with brefeldin A and nocodazole, which inhibits retrograde transport of proteins from Golgi to ER, added to the evidence for the ER as the site of apoB degradation. This conclusion received further support from experiments in which cells were treated with monensin, a Na+ ionophore which halts protein secretion at the level of the trans-Golgi network. Early degradation of nascent apoB (between 10 and 20 min of chase) was observed in monensin-treated cells, but then cellular apoB degradation ceased and apoB was stable during the remaining chase period. More apoB accumulated in the Golgi of cells that had been treated with oleate and monensin. These results suggest that ER degradation occurs in monensin-treated cells, but then stops as apoB is transferred to the Golgi. The results obtained in whole cells were confirmed in studies using isolated ER and Golgi, which indicated that ER contains a proteolytic activity which degrades apoB, in vitro, whereas Golgi does not. ApoB degradation in isolated ER was not reduced by pretreatment with oleate. Finally, protease protection assays carried out with isolated microsomes indicated that a majority of the apoB in both control or oleate-treated HepG2 cells was located on the cytosolic side of the membranes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Glycosylation of P-glycoprotein in a multidrug-resistant KB cell line, and in the human tissues.

P-glycoprotein (P-gp) is thought to transport anti-cancer drugs and to be responsible for the multidrug-resistant (MDR) phenotype. Immunohistochemistry reveals that P-gp is also expressed in normal human tissues, such as the adrenal gland, kidney, liver, and the capillary endothelium of the brain and testis. However, little is known about the structural and functional variations of P-gp in these tissues. With immunoblotting and photoaffinity labeling, we found that the molecular mass of P-gp in these tissues varied between 130-140 kDa. To clarify the post-translational modification of P-gp, we studied the biosynthesis of P-gp in a human multidrug-resistant cell line (KB-C2). We found that P-gp was produced in KB-C2 cells as a 125 kDa precursor and was slowly processed (t1/2 = 45-60 min) to the mature form of 140 kDa. In the presence of tunicamycin, a 120 kDa form of P-gp was synthesized and this form was no longer processed. Treating the 125 kDa precursor form with endo-beta-N-acetylglucosaminidase H (Endo H) and the 140 kDa mature form with N-glycanase diminished the molecular size of P-gp to that of the tunicamycin-treated form. N-Glycanase almost completely removed [3H]glucosamine labeling from P-gp. These data indicate that the major modification of P-gp is N-linked glycosylation. P-gps from KB-C2 cells, kidney and adrenal gland had a different lectin-binding capacity. There seems to be a variety of N-linked glycosylations in tissue and tumor P-gps.     

Schizophrenia, eye movements, and biocultural heterogeneity

Smooth-pursuit eye-tracking dysfunction is a putative genetic trait marker for schizophrenia. In this study 88 Japanese schizophrenics from Kyushu and Okinawa were examined for the marker using precise high-resolution instrumentation: 76% of the schizophrenics from Kyushu and 89% of those from Okinawa had pursuit dysfunction. The presence of the culture-neutral smooth-pursuit marker for schizophrenia in Japan demonstrates that the etic concept "schizophrenia" is cross-culturally valid. Furthermore, the ubiquity of the marker in biologically and culturally diverse populations may indicate a limit on the extent of meaningful heterogeneity likely to be discovered within the condition.     

Pseudomonas putida KF715 bphABCD operon encoding biphenyl and polychlorinated biphenyl degradation: cloning, analysis, and expression in soil bacteria.

We cloned the entire bphABCD genes encoding degradation of biphenyl and polychlorinated biphenyls to benzoate and chlorobenzoates from the chromosomal DNA of Pseudomonas putida KF715. The nucleotide sequence revealed two open reading frames corresponding to the bphC gene encoding 2,3-dihydroxybiphenyl dioxygenase and the bphD gene encoding 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (ring-meta-cleavage compound) hydrolase.     

Characteristics of asparagine-linked sugar chains of sphingolipid activator protein 1 purified from normal human liver and GM1 gangliosidosis (type 1) liver

Asparagine-linked sugar chains of sphingolipid activator protein 1 (SAP-1) purified from normal human liver and GM1 gangliosidosis (type 1) liver were comparatively investigated. Oligosaccharides released from the two SAP-1 samples by hydrazinolysis were fractionated by paper electrophoresis and by Aleuria aurantia lectin-Sepharose and Bio-Gel P-4 (under 400 mesh) column chromatography. Structures of oligosaccharides in each fraction were estimated from data on their effective molecular sizes, behavior on immobilized lectin columns with different carbohydrate-binding specificities, results of sequential digestion by exoglycosidases with different aglycon specificities, and methylation analysis. Sugar chains of SAP-1 purified from normal human liver and from GM1 gangliosidosis (type 1) liver were different from each other, although both of them were derived from complex-type sugar chains. The sugar chains of the former were the following eight degradation products from complex-type sugar chains by exoglycosidases in lysosomes: Man alpha 1----6(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4GlcNAcOT, Man alpha 1----6(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4(Fuc alpha 1----6)GlcNAcOT, Man alpha 1----6Man beta 1----4GlcNAc beta 1----4GlcNAcOT, Man alpha 1----6Man beta 1----4GlcNAc beta 1----4(Fuc alpha 1----6)GlcNAcOT, Man beta 1----4GlcNAc beta 1----4GlcNAcOT, Man beta 1----4GlcNAc beta 1----4(Fuc alpha 1----6)GlcNAcOT, GlcNAc beta 1----4GlcNAcOT, and GlcNAcOT. In contrast to these, the sugar chains of the latter were sialylated and nonsialylated mono- to tetraantennary complex-type sugar chains that were not fully degraded due to a metabolic defect in acid beta-galactosidase activity.     

Streptococcal preparation OK-432 augments cytotoxic activity against an erythroleukemic cell line in humans

Summary The effects of OK-432, an inactivated and lyophilized preparation of a low-virulence strain of Streptococcus pyogenes, were evaluated on the cytotoxicity of lymphoid cells against a natural killer (NK)-sensitive erythroleukemic cell line K562 in patients with malignant diseases. When ten patients were treated postoperatively with daily injections of OK-432, a significant degree of augmentation in the cytotoxicity of peripheral blood lymphoid cells was evoked in all the patients, and the maximum level of cytotoxicity was on the third day after the beginning of the treatment. In spite of the successive daily administration, the level of cytotoxicity declined thereafter, but stayed higher than the pretreatment level. When OK-432 was injected IP in three patients with carcinomatous peritonitis, the cytotoxic activity of ascitic lymphoid cells was significantly enhanced. Cytotoxicity of in vitro-cultured lymphoid cells taken from peripheral blood of normal donors was also augmented by the addition of OK-432.     

Pattern of glycosaminoglycans and glycoproteins associated with nuclei of regenerating liver of rat.

1. Hyaluronic acid was detected as the largest glycosaminoglycan component in the glycosaminoglycan fraction from purified nuclei of regenerating livers as in the case of normal livers (Furukawa, K. and Tarayama, H. (1977) Biochim. Biophys. Acta 499, 278--289). However, the nuclear content of glycosaminoglycans tended to decrease after partial hepatectomy, reaching one-third of the normal liver level at 24--30 h after partial hepatectomy. On the other hand, two new polyanionic components were detected in the glycosaminoglycan fraction from regenerating liver nuclei. 2. One of these new components seems to be a sulfated glycopeptide. The 35SO4 incorporation into this component was stimulated biphasically after partial hepatectomy; the first stimulation occurring immediately after partial hepatectomy and the second stimulation occurring almost in parallel to the DNA synthesis. 3. Another polyacnionic component which also increases in the nuclear content after partial hepatectomy lacks hexuronic acid, sialic acid and 35SO4 and yet it is intensely stained by Alcian Blue. Preliminary investigations revealed the presence of hexose, ribose and phosphate as the major components. 4. In contrast to the primary localization of hyaluronic acid in the chromatin fraction and also in the nonhistone chromosomal protein fraction from it, these new polyanionic components were detected mainly in the karyosol fraction.     

Effect of Chlorine Substitution on the Biodegradability of Polychlorinated Biphenyls

Thirty-one isomers of polychlorinated biphenyl (PCB) were examined for biodegradability by two species of Alcaligenes and Acinetobacter. The following relationships between chlorine substitution and biodegradability of PCBs were observed. (i) Degradation decreased as chlorine substitution increased. PCB isomers containing more than four chlorines were less susceptible to degradation. (ii) PCBs containing two chlorines on either the ortho position of a single ring (i.e., 2,6-) or on both rings (i.e., 2,2′-) showed very poor degradability. (iii) PCBs containing all chlorine atoms on only a single ring were generally degraded faster than when the same number of chlorines were substituted on both rings. (iv) Preferential ring fission of the molecules occurred with nonchlorinated or lesser chlorinated rings. (v) The formation and accumulation of a yellow intermediate was always observed in 4′-chloro-substituted PCBs. (vi) Significant differences between the two organisms with respect to degradability were not observed except for 2,4,6-trichlorobiphenyl.     

Human cytomegalovirus stimulates host cell RNA synthesis.

Human cytomegalovirus infection of human fibroblast cells (WI-38) induced cellular RNA synthesis. The RNA synthesis in infected cultures preceded the synthesis of viral DNA and progeny virus by approximately 24 h. RNA species synthesized in infected cells included ribosomal 28S and 18S; and 4S transfer RNA; all were markedly increased in comparison to uninfected cells. This induction of host cell RNA synthesis was dependent upon a protein(s) that was synthesized during the early stages of infection.