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.     

A naturally occurring mutation of insulin receptor alanine 1134 impairs tyrosine kinase function and is associated with dominantly inherited insulin resistance

We have identified a previously undescribed genetic variant of the insulin receptor (Ala1134----Thr1134) in a family with the Type A syndrome of insulin resistance. Using the polymerase chain reaction to amplify insulin receptor cDNA and genomic DNA (exon 19), this mutation was detected in 1/2 alleles in the proband, her two affected sisters, and her affected father. Two normal alleles were present in the unaffected mother. No additional structural changes were encoded by the remainder of the proband's receptor cDNA. The Ala1134 mutant receptor was expressed in Chinese hamster ovary cells. The expressed mutant receptors were processed normally and displayed normal affinity of insulin binding but were markedly deficient in insulin-stimulated autophosphorylation. The mutant receptor was unable to catalyze the phosphorylation of the endogenous substrate, pp185, and insulin-stimulated kinase activity toward an exogenous substrate in vitro also was markedly impaired. Ala1134 is a highly conserved residue located in a consensus sequence found in most tyrosine kinases. It is likely that this previously uncharacterized residue and/or the immediate region surrounding it are important for normal kinase function in other members of this receptor family. This study also demonstrates that severe insulin resistance with dominant inheritance may be caused by a missense mutation in one allele of the insulin receptor gene.     

Release of heat-labile enterotoxin subunits by Escherichia coli.

Most of the heat-labile enterotoxin (LT) synthesized by Escherichia coli is cell associated; however, a small portion of LT (approximately 10%) is released by bacterial cells into the culture supernatant. The LT subunit B (LT-B) produced by a cloned LT-B gene (tox B) was released in amounts equal to the parent LT release. In contrast, no release of LT subunit A (LT-A) or its smaller derivatives was observed in strains containing cloned toxA genes. The data suggest that LT-B is necessary for the release of LT-A across the bacterial membrane.     

Transfer of the phosphatidyl moiety of phosphatidylglycerol to phosphatidylethanolamine in Escherichia coli

Phosphatidylglycerol was pulse-labeled with radioactive lipid precursors in a serine auxotroph of Escherichia coli. Most of the radioactivity of phosphatidylglycerol labeled in a serine-depleted medium was transferred to phosphatidylethanolamine during a chase in the presence of L-serine, but not in its absence. Metabolism of fatty acyl moieties labeled with [1-14C]acetate, acylated glycerol moieties labeled with [2-3H]glycerol, and phosphate moieties labeled with 32Pi, followed by a chase in the presence of cerulenin, showed that the intact phosphatidyl moiety of phosphatidylglycerol was transferred to phosphatidylethanolamine. The composition of phosphatidylethanolamine molecular species was unaltered and not perturbed by the transfer of the phosphatidyl moiety of phosphatidylglycerol. The increase of phosphatidylethanolamine with a concomitant decrease of phosphatidylglycerol was not coupled with the postulated turnover of phosphatidylglycerol to membrane-derived oligosaccharides and lipoprotein. It is suggested that phosphatidylglycerol is capable of providing its phosphatidyl moiety for the production of phosphatidylethanolamine in response to the relief of serine limitation by addition of L-serine.     

Effects of a triterpenoid saponin on spectral properties of undegraded pea phytochrome

Soyasaponin I, a triterpenoid saponin isolated from etiolated pea (Pisum sativum cv. Alaska) shoots and identified as Pfr killer, was examined for its effects on spectral properties of undegraded pea phytochrome. When soyasaponin I in concentrations of 100 micromolar or lower was added to Pr in the dark, the spectrum of Pr was not significantly affected, whereas in the presence of 120 micromolar or higher concentrations the absorption maximum of Pr shifted from 666 to 658 nanometer with slight decrease of absorbance. After a brief exposure of the mixture to red light, the increase in absorbance at 666 nanometers that occurs in the dark was inhibited at 26 micromolar and higher soyasaponin I concentrations; the maximum effect being reached at about 180 micromolar. The decrease in absorbance at 724 nanometers in the dark after red light irradiation was somewhat inhibited by 60 micromolar and totally prevented by 410 micromolar soyasaponin I. When P₆₅₈ was irradiated with red light in the presence of 220 micromolar or higher soyasaponin I concentrations, a bleached form (P_bl) was produced instead of Pfr. P_bl showed no dark spectral changes, and the phototransformation of P_bl to P₆₅₈ required a significantly high irradiance of far-red light. When the saponin was added to Pfr in the dark, none of the above-described spectral changes occurred, although the same effects were observed after the mixture was exposed briefly to far-red light followed by red light.     

Construction of a physical map of a kanamycin (Km) transposon, Tn5, and a comparison to another km transposon, Tn903

Summary A cleavage map of Tn5, a kanamycin (Km) transposon from plasmid JR67, was constructed from pMKI, a composite plasmid of ColE1 and Tn5, and compared to that of Tn903, a Km transposon from plasmid R6-5. The two transposons showed marked heterogeneity in both the structural gene for Km resistance and the inverted repeat regions as evidenced by their distinctly different restriction maps. This result suggests separate paths of evolution for the two Km transposons.     

Mass spectrometry of trimethylsilyl derivatives of gibberellin glucosides and glucosyl esters

The mass spectra of trimethylsilyl ethers of six gibberellin-β-d-glucopyranosyl ethers and five gibberellin-β-d-glucopyranosyl esters are discussed. The fragmentation patterns are shown to be affected by the structural variations of the aglycones.     

A brief review of bone adaptation to unloading

Weight-bearing bone is constantly adapting its structure and function to mechanical environments. Loading through routine exercises stimulates bone formation and prevents bone loss, but unloading through bed rest and cast immobilization as well as exposure to weightlessness during spaceflight reduces its mass and strength. In order to elucidate the mechanism underlying unloading-driven bone adaptation, ground-based in vitro and in vivo analyses have been conducted using rotating cell culturing and hindlimb suspension. Focusing on gene expression studies in osteoblasts and hindlimb suspension studies, this minireview introduces our recent understanding on bone homeostasis under weightlessness in space. Most of the existing data indicate that unloading has the opposite effects to loading through common signaling pathways. However, a question remains as to whether any pathway unique to unloading （and not to loading） may exist.     

Fluorometric determination of sialic acids using malononitrile in weakly alkaline media and its application to postcolumn labeling in high-performance liquid chromatography

A sensitive method for determination of sialic acids by monitoring the fluorescence produced with malononitrile in borate buffer has been established. Measurement of the fluorescence intensity of the reaction mixture at 430 nm with irradiation at 360 nm allowed determination of 3-60 nmol of sialic acids with high reproducibility. A few amino sugars and deoxy sugars, as well as catecholamines reacted with this reagent; however other carbohydrates, amino acids, amines, aldehydes, and carboxylic acids including alpha-keto acids, etc., showed little reactivity. This method was successfully applied to postcolumn fluorescence labeling of sialic acids in high-performance liquid chromatography.     

Excitability of oxytocin neurons in paraventricular nucleus is regulated by voltage-gated potassium channels Kv4.2 and Kv4.3

Oxytocin is produced by neurons in the paraventricular nucleus (PVN) and the supraoptic nucleus in the hypothalamus. Various ion channels are considered to regulate the excitability of oxytocin neurons and its secretion. A-type currents of voltage-gated potassium channels (Kv channels), generated by Kv4.2/4.3 channels, are known to be involved in the regulation of neuron excitability. However, it is unclear whether the Kv4.2/4.3 channels participate in the regulation of excitability in PVN oxytocin neurons. Here, we investigated the contribution of the Kv4.2/4.3 channels to PVN oxytocin neuron excitability. By using transgenic rat brain slices with the oxytocin-monomeric red fluorescent protein 1 fusion transgene, we examined the excitability of oxytocin neurons by electrophysiological technique. In some oxytocin neurons, the application of Kv4.2/4.3 channel blocker increased firing frequency and membrane potential with extended action potential half-width. Our present study indicates the contribution of Kv4.2/4.3 channels to PVN oxytocin neuron excitability regulation.

Abbreviation: PVN, paraventricular nucleus; Oxt-mRFP1, Oxt-monometric red fluorescent protein 1; PaTx-1, Phrixotoxin-1; TEA, Tetraethylammonium Chloride; TTX, tetrodotoxin; aCSF, artificial cerebrospinal fluid;PBS, phosphate buffered saline 3v, third ventricle.