Structural variations in the glycosaminoglycan-protein linkage region of recombinant decorin expressed in Chinese hamster ovary cells

Decorin is a small flbroblast proteoglycan consisting of a coreprotein and a single chondroitin/dermatan sulfate chain. Thestructure of the carbohydrate-protein linkage region of therecombinant decorin expressed in Chinese hamster ovary cellswas investigated. The decorin was secreted in the culture mediumand isolated by anion-exchange chromatography. The glycosaminoglycanchain was released from the decorin by β-elimination usingalkaline NaBH₄, and then digested with chondroitinase ABC. Thesetreatments resulted in a major and a few minor hexasaccharidealditols derived from the carbohydrate-protein linkage region.Their structures were analyzed by enzymatic digestion in conjunctionwith high-performance liquid chromatography. Two of these compoundshave the conventional hexasaccharide core, HexA1-3GalNAcβ1-4GlcAβ1-3Galβ1-3Galβ1-4Xyl-ol.One is nonsulfated, and the other is monosulfated on C4 of theGalNAc residue. They represent 12% and 60% of the total linkageregion, respectively. The other compound has the hexasaccharidealditol with an internal iduronic acid residue HexA1-3GalNAc(4-sulfate)β1-4IdoA1-3GaIβ1-3Galβ1-4Xyl-ol,which was previously demonstrated in one of the five linkagehexasaccharide alditols isolated from dennatan sulfate proteoglycansof bovine aorta (Sugahara et al, J. Biol Chem., 270, 7204–7212,1995).The compound accounts for 11% of the total linkage region. Thesestructural variations in the linkage hexasaccharide region ofthe decorin strikingly contrast to the uniformity demonstratedin the linkage hexasaccharide structure of human inter--trypsininhibitor (Yamada et al, Glycobiology, 5, 335–341,1995)and urinary trypsin inhibitor (Yamada et al, Eur. J. Biochem.,233, 687–693, 1995), both of which have a single chondroi-tinsulfate chain with a uniform linkage hexasaccharide structure,HexA1-3GalNAc(4-sulfate)β1-4GlcAβ1-3Gal(4-sulfate)β1-3Galβ1-4Xyl,containing a 4-O-sulfated Gal residue. chondroitin sulfate decorin dermatan sulfate glycosaminoglycan proteoglycan     

2(S)-Aminohex-5-ynoic acid,an antimetabolite from Cortinarius claricolor var. Tenuipes

Screening for antimetabolites in edible mushrooms showed that the hot water extract of fruiting bodies of Cortinarius claricolor var. tenuipes strongly inhibited the growth of Bacillus subtilis B-50 in a chemically defined minimal medium. 2(S)-Aminohex-5-ynoic acid was isolated as an active compound.     

Restriction fragment length polymorphism caused by a deletion involving Alu sequences within the human alpha 2-plasmin inhibitor gene

A restriction fragment length polymorphism within the human alpha 2-plasmin inhibitor gene has been detected by Southern blot hybridization using an alpha 2-plasmin inhibitor cDNA probe. This restriction fragment length polymorphism can be attributed to the presence of two alleles, A and B, that are distributed in Hardy-Weinberg equilibrium with frequencies of 73.5% and 2.65%, respectively, in 66 unrelated Caucasian individuals or with frequencies of 51.0% and 49.0%, respectively, in 50 unrelated Japanese individuals. The minor allele, B, is due to a deletion of about 720 base pairs in intron 8 of the alpha 2-plasmin inhibitor gene. Sequence analysis of the deletion junction in allele B and the corresponding regions of allele A demonstrated the presence of oppositely oriented Alu sequences at the 5' and 3' deletion boundaries. These data suggest that this restriction fragment length polymorphism was caused by intrastrand recombination between Alu sequences.     

On the three visual pigments in the retina of the firefly squid,Watasenia scintillans

Summary The deep-sea bioluminescent squid, Watasenia scintillans, has three visual pigments: The major one (A1 pigment) is based on retinal and has _max = 484 nm, the second one (A2 pigment) is based on 3-dehydroretinal and has _max = 500 nm, and the third one (A4 pigment) is based on 4-hydroxyretinal and has _max = 470 nm. The distribution of these 3 visual pigments in the retina was studied by HPLC analysis of the retinals in retina slices obtained by microdissection. It was found that A1 pigment was not located in the specific region of the ventral retina receiving the down-welling light which contains very long photoreceptor cells, forming two strata. A2 and A4 pigment were found exclusively in the proximal pinkish stratum and in the distal yellowish stratum. The role of these pigments in the retina is hypothesized to involve spectral discrimination. The extraction and analysis of retinoids to determine the origin of 3-dehydroretinal and 4-hydroxyretinal in the mature squid showed only a trace amount of 4-hydroxyretinol in the eggs. Similar analysis of other cephalopods collected near Japan showed the absence of A2 or A4 pigment in their eyes.Abbreviations HPLC high-performance liquid chromatography - IS inner segment - OS outer segment     

A possible population monitoring system on environmental mutagens: statistical studies on retinoblastoma in Japan.

Because an increasing number of environmental mutagens have been detected by laboratory screening, the need to monitor human populations directly for mutational risk has been emphasized by many geneticists. They have proposed various monitoring systems but none of them has been practically applied because a large number of samples would need to be studied systematically in such systems. Recent studies by KNUDSON on some malignant tumors give hope that these diseases might be used to assess somatic mutation rates, as suggested by SUTTON (1972). According to KNUDSON, the age of first diagnosis of the diseases may be related to the annual somatic mutation rate. The ages of first diagnosis of unilateral and bilateral retinoblastomas have been compared among four university hospitals in Japan for cases since 1965. Three different types of analysis have been tried: (1) normal distribution; (2) KNUDSON's method; and (3) a modification of VERSCHUER's method for calculating penetration. The age at diagnosis of unilateral cases has fallen in the past ten years. A significant difference in the age of first diagnosis in bilateral retinoblastoma has been found in a hospital in Japan. The changes observed may result from the increased rate of somatic mutations induced by some environmental mutagens.     

Purification and properties of two membrane alkaline phosphatases from Bacillus subtilis 168.

Two alkaline phosphatases were extracted from the membranes of Bacillus subtilis 168 stationary-phase cells and purified as homogeneous proteins by hydroxyapatite column chromatography. Alkaline phosphatases I and II differed in several properties such as subunit molecular weight, substrate specificity, thermostability, Km, pH stability, and peptide maps.     

Identification of NAc-HCPC and NAc-beta-CEC, and qualitative analyses of sulphur amino acids in the urine of a patient with cystathioninuria using liquid chromatography/atmospheric pressure ionization mass spectrometry.

Standard sulphur amino acids and various cystathionine metabolites in the urine of a patient with cystathioninuria were analysed using liquid chromatography/mass spectrometry with an atmospheric pressure ionization interface system. Very intense quasi-molecular ions ([M + H]+) of synthetic cystathionine, N-monoacetylcystathionine, perhydro-1,4-thiazepine-3,5-dicarboxylic acid, S-(3-hydroxy-3-carboxy-n-propyl)cysteine, S-(2-carboxyethyl) cysteine, S-(2-hydroxy-2-carboxyethyl)homocysteine, S-(carboxymethyl)homocysteine, N-acetyl-S-(3-hydroxy-3-carboxy-n-propyl)cysteine and N-acetyl-S-(2-carboxyethyl)cysteine were observed by this method. Quasi-molecular ions ([M + H]+) of these sulphur amino acids were observed in the urine sample of the patient with cystathioninuria, and N-acetyl-HCPC and N-acetyl-beta-CEC as N-substituted sulphur amino acids were also identified in the urine of the same patient.     

pH-dependent membrane fusion activity of a synthetic twenty amino acid peptide with the same sequence as that of the hydrophobic segment of influenza virus hemagglutinin

A twenty amino acid hydrophobic peptide with the same sequence as that of the HA2 N-terminal segment of influenza virus hemagglutinin was synthesized and studied as to its fusion activity. The peptide caused rapid and efficient fusion of egg yolk phosphatidylcholine sonicated vesicles at acidic pH but not at neutral pH. The threshold pH was ca. 6.2 and the maximum fusion occurred at pH 4.8, the half-maximal pH for fusion being 5.6. The pH dependence was similar to that of the parent virus. The fusion efficiency was dependent on the ration of lipid to peptide, increasing with decreasing ratio. The fusion can be rapidly switched on and off by adjusting the pH, to the acidic side and neutral, respectively. The peptide with an acetylated or succinylated N-terminus also showed low pH-induced fusion activity but the pH range was shifted by ca. 1 unit to the acidic side. The results indicate that the HA2 hydrophobic segment in the virus fusion protein is directly involved in the fusion reaction and protonation of the acidic residues in the segment is required for the activity.     

Hereditary alpha 2-plasmin inhibitor deficiency caused by a transport-deficient mutation (alpha 2-PI-Okinawa). Deletion of Glu137 by a trinucleotide deletion blocks intracellular transport

alpha 2-Plasmin inhibitor is the most important physiological inhibitor of fibrinolysis; hence, its deficiency results in a severe hemorrhagic diathesis. We have cloned and characterized a mutant alpha 2-plasmin inhibitor gene from an individual homozygous for the deficiency. By sequencing all the coding exons and exon-intron boundaries of the gene, a trinucleotide deletion in exon VII that results in deletion of Glu137 was identified. We have designated this variant as alpha 2-plasmin inhibitor Okinawa. Using DNA samples amplified with the polymerase chain reaction, hybridization analysis by oligonucleotide probes confirmed the presence of this mutation in all the affected family members, including both parents. To elucidate the mechanism by which this mutation leads to the deficiency, a eukaryotic expression plasmid for alpha 2-plasmin inhibitor containing this mutation was constructed and transfected into COS-7 cells for transient expression analysis. Immunoprecipitation analysis and enzyme-linked immunosorbent assay revealed that the mutant alpha 2-plasmin inhibitor synthesized is mostly retained within the cells as an endoglycosidase H-sensitive form, and only a small portion of it is secreted into the medium as a neuraminidase-sensitive form. These results suggest that the Glu137 deletion leads to the alpha 2-plasmin inhibitor deficiency by causing a block in its intracellular transport from the endoplasmic reticulum to the Golgi complex.