Tissue distribution and quantitation of the mRNAs for three rat tachykinin receptors.

The family of mammalian tachykinin receptors consists of substance P receptor (SPR), neuromedin K receptor (NKR) and substance K receptor (SKR). In this investigation, tissue and regional distributions of the mRNAs for the three rat tachykinin receptors were investigated by blot-hybridization and RNase-protection analyses using the previously cloned receptor cDNAs. SPR mRNA is widely distributed in both the nervous system and peripheral tissues and is expressed abundantly in the hypothalamus and olfactory bulb, as well as in the urinary bladder, salivary glands and small and large intestines. In contrast, NKR mRNA is predominantly expressed in the nervous system, particularly in the cortex, hypothalamus and cerebellum, whereas SKR mRNA expression is restricted to the peripheral tissues, being abundant in the urinary bladder, large intestine, stomach and adrenal gland. Thus, the mRNAs for the three tachykinin receptors show distinct patterns of expression between the nervous system and peripheral tissues. Blot-hybridization analysis in combination with S1 nuclease protection and primer-extension analyses revealed that there are two large forms of SKR mRNA expressed commonly in the peripheral tissues, and two additional small forms of the mRNA expressed specifically in the adrenal gland and eye. These analyses also showed that the multiple forms of SKR mRNA differ in the lengths of the 5' mRNA portions, and that the two small forms of the mRNA, if translated, encode a truncated SKR polypeptide lacking the first two transmembrane domains. This investigation thus provides the comprehensive analysis of the distribution and mode of expression of the mRNAs for the multiple peptide receptors and offers a new basis on which to interpret the diverse functions of multiple tachykinin peptides in the CNS and peripheral tissues.     

Occurrence of D-rhamnan as the common antigen reactive against monoclonal antibody E87 in Pseudomonas aeruginosa IFO 3080 and other strains.

S. Sawada and co-workers reported that a monoclonal antibody (MAb), E87, interacted with about 80% of Pseudomonas aeruginosa isolates, and they separated a rhamnose-rich polysaccharide as the probable antigen for MAb E87 from P. aeruginosa IFO 3080 (S. Sawada, T. Kawamura, Y. Masuho, and K. Tomibe, J. Infec. Dis. 152:1290-1299, 1985). In the present study, the rhamnose-rich polysaccharide was shown to be structurally and immunologically identical to the D-rhamnan of P. aeruginosa IID 1008 (S. Yokota, S. Kaya, S. Sawada, T. Kawamura, Y. Araki, and E. Ito, Eur. J. Biochem. 167:203-209, 1987). Furthermore, a set of enzymes responsible for the formation of GDP-rhamnose (probably in a D-form) from GDP-D-mannose was found in the 100,000 x g supernatant fractions obtained from all of nine P. aeruginosa strains reactive against MAb E87. The result strongly supports a possibility that lipopolysaccharides having a D-rhamnan chain widely occur as the common antigen among various P. aeruginosa isolates.     

Proliferation of peroxisomes and induction of peroxisomal beta-oxidation enzymes in rat hepatoma H4IIEC3 by ciprofibrate

For the analysis of the molecular mechanism of the action of peroxisome proliferators, we attempted to establish the optimal conditions for obtaining the effects of the chemicals in vitro, employing an established cell line, Reuber rat hepatoma H4IIEC3. Histochemical analyses revealed a marked increase in the number, size, and catalase content of peroxisomes in the cells cultured on a medium containing 0.5 mM ciprofibrate, a peroxisome proliferator. The activity of acyl-CoA oxidase, the initial enzyme of the peroxisomal beta-oxidation system, was increased by more than 10-fold by the same treatment. Catalase was also induced significantly, whereas the activities of glutamate dehydrogenase and lactate dehydrogenase, mitochondrial and cytosolic marker enzymes, did not change upon the treatment. Immunoblotting and RNA-blotting analyses confirmed the increases in the amount of protein and mRNA for all the three enzymes of the peroxisomal beta-oxidation system. Cell fractionation experiments gave a partial separation of peroxisomes from other organelles for the induced culture. Thus, H4IIEC3 cells offer a good in vitro model system of the induction of peroxisomes and peroxisomal beta-oxidation enzymes by peroxisome proliferators.     

Increase of a form of UDP-glucuronyltransferase glucuronizing various phenolic xenobiotics and the corresponding translatable mRNA in 3-methylcholanthrene-treated rat liver

Induction of hepatic microsomal UDP-glucuronyltransferase activity toward various phenolic xenobiotics by 3-methylcholanthrene treatment of rats was observed, and the process of the induction was studied. We had previously purified a form of UDP-glucuronyltransferase (called GT-1) having a catalytic activity toward phenolic xenobiotics from liver microsomes of 3-methylcholanthrene-treated rats. The antibodies against GT-1 inhibited the enzyme activity toward those xenobiotics in liver microsomes, and bound to a single protein having a molecular weight of about 54,000 Da (same value as that of GT-1) among microsomal proteins on immunoblotting analysis. The amount of GT-1 protein in hepatic microsomes was found to be increased in close correspondence with the activity increase by 3-methylcholanthrene treatment, by immunoblotting analysis using an uninducible cytochrome P-450 reductase as a negative standard. It was shown by in vitro translation assays that the protein increase described above resulted from the enhancement of the level of translatable mRNA encoding for GT-1. Increases in the amount of the protein immunochemically corresponding to GT-1 in the microsomes from liver of phenobarbital-treated rats and from extrahepatic organs, such as kidney, small intestine, and lung, of phenobarbital- or 3-methylcholanthrene-treated rats were also observed.     

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.