We investigated the carbohydrate structure of the third component of complement (C3) newly synthesized by cultured rat hepatocytes. When the cells were incubated with [3H]mannose, [3H]galactose or [3H]glucosamine, these radioactive precursors were incorporated only into the alpha subunit of C3, demonstrating that only the alpha subunit contains oligosaccharide chains. [3H]Mannose-labelled C3 was purified from the culture medium by immunoaffinity chromatography. Oligosaccharides prepared by Pronase digestion and strong alkaline hydrolysis were separated into two fractions by Bio-Gel P-2 chromatography (Fractions I and II). The two fractions were analysed by concanavalin A-Sepharose chromatography, ion-exchange high-performance liquid chromatography, and Bio-Gel P-4 gel filtration before and after sequential exoglycosidase digestions. It was found that Fraction I contained two complex type oligosaccharide chains, (NeuAc)2(Gal)2(GlcNAc)2(Man)3(GlcNAc)2 and (NeuAc)3(Gal)3(GlcNAc)3(Man)3(GlcNAc)2, and Fraction II contained the high-mannose type, consisting mainly of (Man)8(GlcNAc)2. Taken together with the carbohydrate composition of rat serum C3, the results suggest that rat C3 has one high-mannose type oligosaccharide chain and two complex type chains in the alpha subunit, which is different from the proposal for human C3.

Animals↗

PubMed1986

Structural analysis of the asparagine-linked oligosaccharides of rat haptoglobin metabolically labeled in a hepatocyte culture system.

We analyzed the asparagine-linked oligosaccharide chains of rat haptoglobin which were synthesized and secreted by hepatocytes in primary culture. When the cells were incubated with either [3H]mannose, [3H]galactose, or [3H]fucose, all the radioactive precursors were incorporated into the beta subunit of haptoglobin. [3H]Mannose-labeled haptoglobin was purified from the culture medium by immunoaffinity chromatography, and [3H]oligosaccharides were prepared by strong alkali-borohydride treatment. The oligosaccharides obtained were analyzed by anion-exchange high-performance liquid chromatography, concanavalin-A--Sepharose chromatography and Bio-Gel P-4 chromatography before and after sequential exoglycosidase digestions. The oligosaccharides labeled with [3H]fucose or [3H]galactose were also characterized by the above methods. The results indicate that rat haptoglobin contains two complex-type oligosaccharide chains in each beta subunit; one with a possible structure of ( +/- NeuAc----Gal beta----GlcNAc beta----)3(Man alpha----)2 Man beta----GlcNAc----( +/- Fuc alpha----)GlcNAc and the other with ( +/- NeuAc----Gal beta----GlcNAc beta----Man alpha----)2 Man beta----GlcNAc----( +/- Fuc alpha----)GlcNAc.

Animals↗

PubMed1986

Preliminary crystallographic study of a ribulose-1,5-bisphosphate carboxylase-oxygenase from Chromatium vinosum.

Crystals of a ribulose-1,5-bisphosphate carboxylase-oxygenase from Chromatium vinosum were obtained with the hanging-drop vapor diffusion technique, using polyethylene glycol 4000 as precipitant. The crystal belongs to the cubic system, space group I432, with unit cell dimension a = 245.9 A. An asymmetric unit includes one-quarter (L2S2, L: large subunit, S: small subunit) of a hexadecameric molecule (L8S8, 544,000 Mr), which is located on the crystallographic point symmetry 222 or 4. The crystal diffracts to at least 3.0 A resolution.

Chromatium↗

PubMed1986

Crystallization and preliminary X-ray diffraction study of ferrocytochrome c2 from Rhodopseudomonas viridis.

Crystals of ferrocytochrome c2 from a non-sulphur purple photosynthetic bacterium, Rhodopseudomonas viridis, have been grown from ammonium sulphate solution at pH 8.5 by the sitting-drop vapour-diffusion procedure. The crystals belong to the trigonal system, space group P3(1)21 (or its enantiomorph P3(2)21) with unit-cell dimensions of a = b = 75.8 A and c = 40.1 A, and diffract to at least 2.0 A resolution. Assuming that an asymmetric unit contains one protein molecule (approx. 12,300 Mr), the solvent content of the crystal is approximately 54.5% (v/v).

Crystallization↗

PubMed1986

Novel blockade by brefeldin A of intracellular transport of secretory proteins in cultured rat hepatocytes.

We examined the effect of brefeldin A, an antiviral antibiotic, on protein synthesis, intracellular processing, and secretion in primary culture of rat hepatocytes. The secretion was strongly blocked by the drug at 1 microgram/ml and higher concentrations, while the protein synthesis was maintained fairly well. Pulse-chase experiments with [35S]methionine demonstrated that brefeldin A completely blocked the proteolytic conversion of proalbumin to serum albumin up to 60 min of chase, although its conversion was observed as early as 20 min in the control cells. The drug also inhibited the terminal glycosylation of oligosaccharide chains of alpha 1-protease inhibitor and haptoglobin. These two modifications have been shown to occur at the trans region of the Golgi complex. The drug, however, had no effect on the proteolytic processing of the haptoglobin proform which takes place within the endoplasmic reticulum. Such an effect by brefeldin A is very similar with that induced by the carboxylic ionophore monensin. However, in contrast to evidence that monensin causes a delayed secretion of the unprocessed forms of these proteins, brefeldin A allowed the completely processed forms to be secreted after a prolonged accumulation of the unprocessed forms. Morphological observations demonstrated that the endoplasmic reticulum was markedly dilated by treatment with the drug at 10 micrograms/ml which continuously blocked the secretion. On the other hand, brefeldin A caused no inhibitory effect on the endocytic pathway as judged by cellular uptake and degradation of 125I-asialofetuin. These results indicate that brefeldin A is a unique agent which primarily impedes protein transport from the endoplasmic reticulum to the Golgi complex by a mechanism different from those considered for other secretion-blocking agents so far reported.

Adenosine Triphosphate↗

PubMed1986

Density-of-states investigation of C8K and occurrence of the interlayer band.

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Journal Article↗

PubMed1986

[Determination of thyroid volume using computerized ultrasonography and its clinical application].

A new method for determining thyroid gland volume was developed by means of computerized ultrasonography. A maximum area of the thyroid (S) was obtained from coronal sections and a maximum thickness (C) was obtained from sagittal sections. The thyroid volume was then calculated by SXC. The volume of phantoms and pig thyroids obtained by this method was identical with the actual volume. The thyroid volume in this method correlated well with the estimated volume in patients with Graves' disease undergoing thyroidectomy (r = 0.954). The measurement was then performed in 56 healthy subjects (male 28, female 28, age 15-68 y.o.) and 126 patients with thyroid diseases. The thyroid volume in healthy men was significantly larger than that in women (18.4 +/- 6.1 ml vs 13.9 +/- 4.9 ml, Mean +/- SD, p less than 0.01). The thyroid volume in normal subjects correlated with body height, body weight or body surface area (p less than 0.01), which may explain the sex differences. An attempt was made to calculate the prospective thyroid volume (Y ml) from body height as follows; Y = 0.425 X - 52.8, X; body height (cm). Four out of thirty patients with untreated Graves' disease had a gland volume of the normal range, but in all patients the thyroid volume was larger than the prospective volume. The thyroid volume and the rate of volume increase (determined thyroid volume/calculated prospective volume) were correlated with the serum levels of T3, T4 concentration in the untreated stage. The volume and the rate of volume increase did not correlate with serum TSH receptor antibody activity. The thyroid volume in patients with Graves' disease who had been in remission (33.0 +/- 13.5 ml) was significantly smaller than that in untreated patients (51.8 +/- 24.4 ml, p less than 0.02). These findings indicate that the present method is reliable and clinically useful for the investigation of thyroid diseases.

Adolescent↗

PubMed1986

An intravitreal cannula system: long-term follow-up study.

An intravitreal cannula for chronic drug delivery was implanted in the eyes of two rabbits and one monkey. The rabbits were followed up for three years after surgery and the monkey was followed up for two and one-half years. Clinical observations did not reveal any adverse effects from the cannula implantation. Patency of the cannula was demonstrated by using sodium fluorescein. Histopathological studies with light and electron microscopy revealed the scar tissue around the cannula to be minimal and localized to the wound site. The optic nerve and the retina and choroid at the posterior pole did not show any abnormalities. These results suggest that this system can be used for repeated or continuous drug delivery to the vitreous over a long period of time in experimental models.

Animals↗

PubMed1986

Role of molecular oxygen in lignin peroxidase reactions.

Homogeneous lignin peroxidase (diarylpropane oxygenase) oxidized veratryl alcohol to veratryl aldehyde under anaerobic conditions in the presence of either H2O2, m-chloroperoxybenzoic acid (mCPBA), or p-nitroperoxybenzoic acid (pNPBA). Lignin peroxidase also oxidized the 1-(3',4'-diethoxyphenyl)-1,2-dihydroxy-(4"-methoxyphenyl)-propane I under anaerobic conditions in the presence of mCPBA to yield 3,4-diethoxybenzaldehyde III and 1-(4'-methoxyphenyl)-1,2-dihydroxyethane IV. In contrast to what occurs under aerobic conditions, under anaerobic conditions no 2-hydroxy-1-(4'-methoxyphenyl)-1-oxoethane V was obtained. During the diarylpropane I cleavage under anaerobic conditions, 18O from H2(18)O was incorporated into the alpha-position of the phenylglycol IV. Lignin peroxidase also hydroxylated 1-(4'-ethoxy-3'-methoxyphenyl)propane II at the alpha-position to yield 1-(4'-ethoxy-3'-methoxyphenyl)-1-hydroxypropane VI under anaerobic conditions in the presence of mCPBA. During the phenylpropane II hydroxylation under anaerobic conditions, 18O from H2(18)O was incorporated into the alpha-position of VI. These results are rationalized according to a mechanism involving an initial one-electron oxidation of the diarylpropane I by the lignin peroxidase compound I to form a benzene pi cation radical which undergoes alpha, beta cleavage to produce a benzaldehyde and a C6C2 benzylic radical. The latter is then attacked by O2 to form a hydroperoxy radical which may decompose through a tetroxide to form the phenylglycol IV and phenylketol V. Under anaerobic conditions the C6C2 benzylic radical is probably oxidized to a carbonium ion which would be subsequently attacked by H2O to yield the phenylglycol V.

Anaerobiosis↗

PubMed1986

Crystal structures of modified myoglobins. I. Heme orientation and structural changes around heme in myoglobins reconstituted with isopemptoheme, pemptoheme, 2-ethyldeuteroheme, and 4-ethyldeuteroheme.

The crystal structures of sperm whale metmyoglobins reconstituted with four modified hemes, isopemptoheme, pemptoheme, 2-ethyldeuteroheme, and 4-ethyldeuteroheme, have been determined and refined at 2.2 A resolution to R = 0.217, 0.218, 0.213, and 0.222, respectively. All the crystals of these myoglobins are isomorphous with that of native metmyoglobin. The structural changes of the modified myoglobin from the native myoglobin were examined on difference Fourier maps; the orientation of 4-ethyldeuteroheme in the heme pocket is such that the heme is rotated by 180 degrees about an axis through the alpha-gamma-meso carbons, whereas the orientations of the other three hemes are the same as that of the protoheme in the native myoglobin. The changes of the structures around the heme become greater in the order of isopemptoheme, 2-ethyldeuteroheme less than pemptoheme less than 4-ethyldeuteroheme. The magnitudes of the changes seem to be related to the oxygen affinities of these four reconstituted myoglobins.

Animals↗

PubMed1986

Crystal structures of modified myoglobins. II. Relation between oxygen affinity properties and structural changes around heme in myoglobins reconstituted with 2,4-diisopropyldeuteroheme, 2-isopropyl-4-vinyldeuteroheme, and 2-vinyl-4-isopropyldeuteroheme.

The crystal structures of sperm whale metmyoglobins reconstituted with three kinds of modified hemes, 2,4-diisopropyldeuteroheme, 2-isopropyl-4-vinyldeuteroheme, and 2-vinyl-4-isopropyldeuteroheme, have been determined and refined at 2.2 A resolution to R = 0.216, 0.219, and 0.195, respectively. All the crystals of these myoglobins are isomorphous with that of native metmyoglobin. The 2-vinyl-4-isopropyldeuteroheme was found to be in a reverse orientation, in which the heme plane is rotated by 180 degrees about an axis through the alpha-gamma-meso carbons, whereas the orientations of the other two hemes were the same as that of protoheme in native myoglobin. In the myoglobins with 2,4-diisopropyldeuteroheme and 2-vinyl-4-isopropyldeuteroheme, both of which have lower oxygen affinities than native myoglobin, the bulky isopropyl side chain pushes Phe 43 0.7 A toward His 64 (the distal histidine) in the former, and the whole E helix at most 1.5 A, including a 0.7 A shift of the His 64 imidazole ring, in the latter. The changes of the structures prevent His 64 from forming a hydrogen bond with the liganded oxygen molecule, so that these two modified myoglobins show low oxygen affinities. On the other hand, there is no such drastic displacement in myoglobin with 2-isopropyl-4-vinyldeuteroheme, which has a slightly higher oxygen affinity than native myoglobin.

Animals↗