Isolation and characterization of major urinary amino acid O-glycosides and a dipeptide O-glycoside from a new lysosomal storage disorder (Kanzaki disease). Excessive excretion of serine- and threonine-linked glycan in the patient urine

Four major sialo compounds, termed GP-M1, GP-D1, GP-D2, and GP-D3 have been isolated from the urine of a novel glycoprotein storage disorder patient with angiokeratoma corporis diffusum which was discovered by Kanzaki et al. (Kanzaki, T., Yokota, M., Mizuno, N., Matsumoto, Y., and Hirabayashi, Y. (1989) Lancet April 22, 875-877). Based on the results of fast atom bombardment mass spectrometry, methylation analysis, and proton nuclear magnetic resonance spectroscopy, their chemical structures were concluded to be: (formula; see text) The yields of GP-M1, GP-D1, GP-D2, and GP-D3 were approximately 15, 6, 50, and 5 mg/liter of urine, respectively. The most major compound GP-D2, was further purified into single molecular species, threonine and serine type, by reversed phase high performance liquid chromatography. NMR analysis of the two purified compounds with single molecular species showed that the chemical shifts of anomeric protons of GalNAc were significantly different between threonine- and serine-linked GalNAc. Neither mannose-containing glycopeptides nor glycosphingolipids were excreted in the patient urine. From these results, this disease is thought to be caused by the deficiency of a lysosomal enzyme(s) acting on O-linked glycan chains.     

Human serum deoxyribonuclease I (DNase I) polymorphism: pattern similarities among isozymes from serum, urine, kidney, liver, and pancreas.

We have devised a zymogram method with high sensitivity and resolution for investigating molecular heterogeneity and genetic polymorphism of deoxyribonuclease I. A combination technique of polyacrylamide-gel isoelectric-focusing electrophoresis and the newly developed zymogram method have led to the discovery of genetic polymorphism of human serum DNase I. Family studies showed that the three common phenotypes--DNASE1 1, DNASE1 1-2, and DNASE1 2--and the other five relatively rare phenotypes--DNASE1 1-3, DNASE1 2-3, DNASE1 2-4, and DNASE1 3-4--represent homozygosity or heterozygosity for four autosomal codominant alleles, DNASE1 *1, DNASE1 *2, DNASE1 *3, and DNASE1 *4. The frequencies of DNASE1 *1, DNASE1 *2, DNASE1 *3, and DNASE1 *4 calculated in a Japanese population were .5517, .4358, .0104, and .0021, respectively. Moreover, it was found that urine and extracts of kidney, liver, and pancreas, as well as serum, can be used for DNase I phenotyping.     

Is the IS1-flanked r-determinant of the R plasmid NR1 a transposon?

The 23 kilobase multiple drug resistance r-determinant (r-det) of the R plasmid NR1 is an IS1-mediated transposon, Tn2671. Drug-resistant Escherichia coli transductants isolated after infection with bacteriophage P1::Tn2671 derivatives carry the intact r-det in their chromosomes. Independently isolated transductants carry the r-det at different locations on the chromosome. From the E. coli chromosome, Tn2671 can transpose to various locations on the phage P7 genome. Throughout these processes, r-det is maintained as a stable unit. Various possible molecular mechanisms, which all might contribute with characteristic frequencies to the transposition of Tn2671, are discussed. The results presented are relevant to the understanding of mechanisms for a wide spreading of drug resistance genes.     

Seven sesquiterpene lactones from Inula britannica var. chinensis

Four known sesquiterpene lactones, tomentosin, ivalin, 4-epi-isoinuviscolide and gaillardin, together with three new lactones, inuchinenolides A, B and C, were identified in the whole plant of Inula britannica var. chinensis.     

On the role of IS1 in the formation of hybrids between the bacteriophage P1 and the R plasmid NR1

Summary The genomes of bacteriophage P1 derivatives carrying drug resistance genes derived from an R plasmid NR1 were analysed by restriction cleavage and be DNA-DNA hybridization. Two representatives of a class of oversized P1CmSmSu phages were identified as P1 carrying the entire r-determinant of NR1 together with its two flanking, directly repeated IS1. In one case the r-determinant insertion is carried at the site of the residential IS1 of P1, in the other case it is transposed into another region of the P1 genome. Models postulate that the first type resulted from reciprocal recombination within IS1 elements and that the formation of the second type of P1-R hybrid depended both on IS1 mediated transposition and reciprocal recombination. Plaque forming P1Cm or P1CmSm phages are explained as IS1 mediated deletion derivatives of P1CmSmSu, although an alternative model postulates that sometimes P1Cm phages might result from two consecutive transposition events of only one IS1 without involving reciprocal recombination. Secondary P1 derivatives carrying only one IS1 at the site of the original r-determinant or of Cm insertions into P1 must have been produced by reciprocal recombination between the two IS1 flanking the insertions. An implication from this study, that any genetic material carried adjacent to an IS1 element may undergo passive transposition, is discussed.     

Calcium binding of troponin C. I. A potentiometric titration study.

Structural changes of troponin C on calcium binding were studied by hydrogen ion titration, circular dichroism, and fluorescence measurements. The potentiometric titration curves in the carboxyl region are shifted towards lower pH with calcium binding. The intrinsic pK of the carboxyl groups at the calcium binding sites decreases by 0.8 pK unit on calcium binding; on the other hand, magnesium ions have little effect on the intrinsic pK of the carboxyl groups. The intrinsic pK of the imidazole group is not affected by calcium binding. The value of w, an electrostatic interaction factor, is identical for calcium-free and calcium-bound troponin C and is about half of the value calculated assuming a compact sphere. The results of difference titration on the calcium binding indicate that the pH of troponin C solution increases on addition of CaCl2 up to 2 mol of Ca2+ per mol of troponin C and then decreases on further addition of CaCl2. The pH increase is depressed in the presence of MgCl2, in the low pH region, or at high ionic strength. The pH increase is also observed on addition of MgCl2. The ellipticity at 222 nm was measured under the same conditions as the difference titration measurements, and the relation between the pH change and the conformational change of troponin C on calcium binding is discussed based on the results obtained. The number of calcium binding sites and the binding constants estimated by analysis of these difference titration curves were in agreement with the results of Potter and Gergely (22). No magnesium binding site was observed. The tyrosine fluorescence measurements indicated that the binding site near tyrosine-109 is one of the high affinity sites.     

Microbiological production of radioisotopically labelled 16 alpha-hydroxylated steroids in trace quantities.

[14-14C]16 alpha-Hydroxy-C-18- and C-19-steroid hormones were obtained in good yields by microbiological hydroxylation of correspondingly labelled steroids by Streptomyces roseochromogenes NRRL B-1233. Trace quantities of the labelled substrates were incubated on a rotary shaker (220 rpm) at 27 degrees C. The radioactive products were chromatographically separated, identified and the radiochemical purity was established by isotopic dilution analysis. The specific activities of 16 alpha-hydroxy-steroids obtained were assumed to be the same as those of the substrates, namely, 57.5 mCi/mmole for 16 alpha-hydroxy-4-androstene-3,17-dione, 57.5 mCi/mmole for 5-androstene-3 beta,16 alpha,17 beta-triol, 57.5 mCi/mmole for 16 alpha-hydroxy-dehydroepiandrosterone, 55.7 mCi/mmole for 16 alpha-hydroxy-estrone, and 57.5 mCi/mmole for 16 alpha-hydroxy-testosterone.     

Biochemical studies on abnormal erythrocyte membranes protein abnormality of erythrocyte membrane in biliary obstruction

Biochemical studies on erythrocyte membranes from eleven obstructive jaundice patients (due to various disorders) have been undertaken. By scanning electron microscopic observation these erythrocytes were spur and target in appearance. The lipid composition showed a marked increase in both cholesterol and phosphatidylcholine. In addition to these changes, it was unexpectedly demonstrated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate that a specific membrane protein component 4.2 was reduced or absent in all cases tested. This membrane protein abnormality was identical with that of hereditary spherocytosis erythrocyte membranes. It is of particular interest to note that after surgical relief of biliary obstruction in a typical case of common duct cholelithiasis, the disc electrophoretic pattern of erythrocyte membranes became normal and both lipid composition and red cell morphology returned to normal.     

Purification of host DNA synthesis-suppressing factor (DSF) produced by infection with measles virus.

Host DNA synthesis-suppressing factor (DSF) produced into culture fluid of cloned HeLa cells (HeLa C-9) infected with a small plaque variant of Toyoshima strain of measles virus was purified by precipitation with ammonium sulfate, chromatography on CM-cellulose and DEAE-cellulose, and gel-filtration on Sephadex G-100 and G-200. The specific activity of the finally purified DSF was 302 units/mg of protein representing approximately 300-fold purification. The molecular weight of DSF was estimated to be about 55 000. By isoelectric focusing, two kinds of DSF having isoelectric points of 4.24 and 5.24 were detectable. The purified DSF was able to suppress host DNA synthesis of HeLa cells, continuous human lymphoid cells (NC-37), mouse L cells and Meth-A cells derived from an ascitic tumor of the mouse. The activity of the purified DSF was inactivated by heating at 56 C for 30 min or by treatment with trypsin.