Fucosylation of membrane proteins in soybean cultured cells : effects of tunicamycin and swainsonine

Cultures of soybean cells incorporate [5,6-³H]-l-fucose into various cellular components including lipids and proteins. The membrane glyco-proteins were digested with pronase to produce glycopeptides, and the glycopeptides were isolated on columns of Biogel P-4. The major fucoselabeled glycopeptide sized as a Hexose_15-17-N-acetylglucosamine₂ (GlcNAc₂) on columns of Biogel P-4. Fucose incorporation was also examined in the presence of the processing inhibitor swainsonine, and the glycosylation inhibitor tunicamycin. In the presence of swainsonine, the incorporation of fucose was not reduced but the glycopeptides were smaller in size and migrated like Hexose_12-13-GlcNAc₂ structures. On the other hand, tunicamycin inhibited the incorporation of fucose into the glycopeptides by 70 to 80%, indicating that the l-fucose was present in N-linked oligosaccharides.     

The ultrastructure of mitosis inIsochrysis galbana parke (Prymnesiophyceae)

Summary Mitosis and cytokinesis have been studied in the flagellate algaIsochrysis galbana Parke (Prymnesiophyceae). Nuclear division is preceded by replication of the flagella and haptonema, the Golgi body and the chloroplast; fission in the chloroplast occurs in the region of the pyrenoid. During prophase, spindle microtubules radiating from two ill-defined poles are formed. The nuclear envelope breaks down and the chromatin condenses. At metaphase the spindle is fully developed, some pole-to-pole microtubules passing through the well-defined chromatin plate, others terminating at it. No kinetochores or individual chromosomes were observed. By late metaphase, many Golgi-derived vesicles may be seen against the two poleward faces of the metaphase plate. During anaphase, the two daughter masses of chromatin move towards the poles. In early telophase, the nuclear envelope of each daughter nucleus is complete only on the side towards the adjacent chloroplast, remaining open on the interzonal side. However, during telophase each nucleus becomes reorientated so that it lies lateral to the long axis of the spindle and with its open side towards the chloroplasts. By late telophase, each new nuclear envelope is complete and confluence with the adjacent chloroplast ER established.Cytokinesis and subsequent segregation of the daughter cells are effected by the dilation of Golgi- and ER-derived vesicles in the interzonal region. No microtubular structures are involved. Comparisons with the results from other studies of mitosis in members of thePrymnesiophyceae show that they all have a number of features in common, but that there are differences in detail between species.     

Human aldolase isozyme gene: the structure of multispecies aldolase B mRNAs.

A complete nucleotide sequence of human aldolase B mRNA was determined with a recombinant cDNA (pHABL120-3). The cDNA insert was composed of 1,652 bases excluding poly(A) tail and the sequence was consistent with the previous results reported by others. However, S1 nuclease mapping and subsequent genomic analysis allowed us to know that the clone possesses two more sites corresponding to 5'-termini in the 5'-noncoding region and another site of polyadenylation in the 3'-noncoding region. In fact, the major aldolase B mRNA species occupying 90% of the total mRNAs initiated at the predominant position corresponding to the position around -82 of the 5'-noncoding sequence in pHABL120-3 and terminated at the distal polyadenylation site. Second species accounting for 9% of the mRNAs initiated at the same site and terminated at the proximal polyadenylation site. The remainings have a longer 5'-noncoding sequence which starts from further upstream region of the major one and pHABL120-3 corresponds to one of these largest clones.     

Neutral glycosphingolipids of ova of the fresh-water bivalve, Hyriopsis schlegelii

The neutral glycosphingolipids of ova of the fresh-water bivalve, Hyriopsis schlegelii were characterized. The most abundant glycolipid was ceramide monosaccharide, followed by ceramide trisaccharide, ceramide tetrasaccharide, and ceramide disaccharide. More complex neutral glycolipids accounted for almost one-third of the total. The total amount of these glycolipids was 0.59 mg/g of dry weight of the ova preparation, a yield which was one-seventh of that of spermatozoa neutral glycolipids. Structural analyses were performed by enzymatic hydrolysis of the glycolipids with exoglycosidases, permethylation experiments, and also immuno-chemical assays. The proposed structures are as follows: ceramide monosaccharides, Gal-Cer and Glc-Cer; ceramide disacharides, Gal(beta 1-4)Gal-Cer, Gal(beta 1-4)Glc-Cer, and Man(beta 1-4)Glc-Cer; ceramide trisaccharide, Man(alpha 1-3)Man(beta 1-4)Glc-Cer; ceramide tetrasaccharides, Man(alpha 1-3)[Xyl(beta 1-2)]Man(beta 1-4)Glc-Cer, GlcNAc(beta 1-2)Man(alpha 1-3)Man(beta 1-4)Glc-Cer, Man(alpha 1-3)[Gal(beta 1-2)]Man(beta 1-4)Glc-Cer, and Man(alpha 1-2?)Man(alpha 1-3)Man(beta 1-4)Glc-Cer. The latter two ceramide tetrasaccharides were new types of glycosphingolipids. The spectrum of ova glycolipids appeared to be more complicated than that of the spermatozoa glycolipids. The ova glycolipids characterized here, with the exception of ceramide tetrasaccharides, contained considerable amounts of 2-hydroxy fatty acids, which were not observed in the spermatozoa glycolipids. The major sphingosine base was C18-sphingenine in all the ova glycolipids as well as in the spermatozoa glycolipids. However, the content of anteiso type of sphingosine base was 2- to 3-fold higher in the ova than in the spermatozoa.     

Alterations in Fc receptor function of macrophages from streptozotocin-induced diabetic rats

The presence of elevated levels of circulating immune complexes in diabetic humans and animals suggests impaired phagocyte function. To evaluate FcR-mediated phagocytosis, resident peritoneal macrophages were harvested from control, streptozotocin-induced diabetic, and insulin-treated diabetic rats. FcR number and avidity were determined from Scatchard analysis of binding of 125I-labeled aggregated rat IgG (ARG) to macrophages. The total and fractional catabolic capacity were determined by quantitating the digestion of ARG as a percent of the total ARG added and as a percent of ARG bound. Insulin-deficient diabetic rats had an increase in the number of FcR per cell (26.8 +/- 3.5 X 10(4)) as compared with control animals (13.1 +/- 1.2 X 10(4)) (p less than 0.01). In contrast, insulin-treated diabetic animals had a reduction in the number of FcR per cell (9.8 +/- 1.4 X 10(4)) (p less than 0.01). FcR of macrophages from insulin-deficient diabetic rats had a lower avidity (Kd = 6.9 +/- 1.8 X 10(-10)M) when compared with control (3.7 +/- 0.6 X 10(-10)M) and insulin-treated diabetic rats (3.6 +/- 0.9 X 10(-10)M) (p less than 0.01). Total catabolism of ARG by macrophages from both insulin-deficient and insulin-treated diabetic rats was reduced (31.0% +/- 3.4 and 17.5% +/- 3, respectively) when compared with controls (49.6% +/- 5.2) (p less than 0.01). Fractional catabolism by macrophages from insulin-deficient diabetic rats was significantly reduced (21% +/- 1.9 and 4.6% +/- 0.9/10(4) FcR) when compared with results from control rats (26% +/- 1.3 and 6.7% +/- 0.7/10(4) FcR) (p less than 0.01), whereas the results from insulin-treated diabetic rats (32% +/- 2.4 and 10.8% +/- 1.0/10(4) FcR) (p less than 0.01) were greater than those from controls. These studies demonstrate that FcR-mediated phagocytosis of soluble, "model" immune complexes is impaired in macrophages from both insulin-deficient and insulin-treated diabetic rats; however, different mechanisms account for this impairment in phagocytosis. Despite an increase in FcR number of macrophages from insulin-deficient diabetic rats, the depression of post-receptor-mediated catabolism results in a net depression in phagocytic activity. In contrast, macrophages from insulin-treated diabetic rats display augmented post-receptor-mediated catabolism; however, this does not overcome the low initial binding of ARG to the cell that results from the depression of FcR number.     

Effect of high carbohydrate diet on serum gamma-glutamyl transpeptidase fraction pattern in Japanese young men

Changes in the activity of serum gamma-glutamyl transpeptidase (gamma-GTP) and the percentage of the gamma-GTP fraction in healthy young men given a high carbohydrate diet (480-636 g/day, 80% of the total energy) for 21 days were examined. Serum total gamma-GTP activity showed no significant change in four healthy young volunteers who received high carbohydrate diet for 21 days. However, the percentage of the gamma-GTP (1) fraction increased significantly (P less than 0.01) from the basal level of 55.6 +/- 4.0% to 67.6 +/- 0.9% on day 10, and then decreased to 58.4 +/- 1.4% on day 21. When the experimental diet was replaced by usual diet, the percentage of the gamma-GTP (1) fraction returned to the same level as before the experiment. It is concluded from the results that the nutrient intake affects the percentage of gamma-GTP (1), but not the total serum gamma-GTP activity.     

Freeze-fracture studies of the sinoatrial and atrioventricular nodes of the caprine heart,with special reference to the nexus

Summary The caprine sinoatrial node (SAN) and atrioventricular node (AVN) were studied by freeze-fracture techniques, and their nexus or gap junction structure were compared with that of ordinary atrial and ventricular muscle cells. The general features of the nexus in both the SAN and AVN were essentially identical. Approximately two-thirds of the nexuses observed in the nodal cells consisted of typical macular arrangements of nexal particles, and the remaining third, of atypical configurations of either circular arrangements or linear arrays of particles in continuity with the macular nexuses. Such atypical nexuses were never observed in the ordinary adult myocardial cells. Quantitative analysis revealed that all of the nexuses in the nodal cells measured, were less than 0.1 m², whereas the majority of the nexuses in ordinary myocardial cells (64% in the atrium and 76% in the ventricle) were larger than 0.1 m². No significant differences in diameter and center-to-center distance of nexal particle were found between the nodal cells and ordinary myocardial cells.     

Conversion of 4-aminobutyraldehyde to γ-aminobutyric acid in striatum treated with semicarbazide and kainic acid

4-Aminobutyraldehyde (ABAL) has been shown to cross the blood-brain barrier and to be converted rapidly to -aminobutyric acid (GABA) in various regions of the brain. In this paper, the formation of GABA from ABAL was studied with striatum that had suffered a lesion to GABA synthesis via glutamic acid decarboxylase (GAD). The GABA formation from ABAL was invariably observed in striatum in which GAD was severely inhibited by semicarbazide or kainic acid. Thus, this is another pathway for GABA formation.     

Analysis of powder and single crystal electron paramagnetic resonance spectra of deoxy myoglobins containing cobalt (II) proto-and mesoporphyrins IX at various microwave frequencies

Artificial myoglobins (Mbs) substituted for protoheme with Co(II) proto-and mesoporphyrins IX (proto-and meso-CoMbs, respectively) were prepared. The principal values and eigenvectors of g tensors and the hyperfine coupling tensors of the paramagnetic Co(II) centers of their deoxy forms have been determined by single crystal EPR spectroscopy at 77 K in order to elucidate orientation and electronic structure of the prosthetic group in myoglobin. The orientation of the porphyrin plane of deoxy meso-CoMb were found to be identical to that of deoxy proto-CoMb. However, the in-plane hyperfine coupling constants of deoxy meso-CoMb were more anisotropic and larger than those of deoxy proto-CoMb, suggesting an increase in the electron spin density on the Co(II) ion upon the exchange of protoporphyrin IX with mesoprophyrin IX. Powder EPR spectra of these CoMbs, which were measured at S- and L-band microwave frequencies, exhibited well resolved 59Co hyperfine splittings and can be clearly interpreted by the use of the EPR parameters obtained from single crystal EPR measurements.