Effects of a newly synthesized leukotriene antagonist, NZ-107, on immediate-type hypersensitivity reaction in rats and guinea-pigs.

The effects of 4-bromo-5-(3-ethoxy-4-methoxybenzylamino)-3(2H)-pyridazinone (NZ-107) on immediate type hypersensitivity reactions in rats and guinea-pigs were studied. 1. When NZ-107, at a dose of 50 mg/kg (i.p.) or 100 mg/kg (orally), was administered to rats, 48-h homologous passive cutaneous anaphylaxis (PCA) reaction and histamine-, leukotriene C4 (LTC4)- and leukotriene D4 (LTD4)-induced skin reactions were suppressed by the agent. 2. NZ-107 (10(-6) g/ml) inhibited both LTC4- and LTD4-induced contractions of isolated rat stomach smooth muscle. 3. NZ-107 inhibited antigen-induced histamine release from rat peritoneal mast cells by 26% at a concentration of 10(-4) g/ml. 4. NZ-107, at doses of 25 and 50 mg/kg (orally), significantly inhibited guinea-pig 3-h heterologous PCA reaction. 5. NZ-107 inhibited antigen-induced histamine release from guinea-pig lung tissue by 17% and 48% at concentrations of 5 x 10(-5) and 10(-4) g/ml, respectively. 6. NZ-107, at doses of 25 and 50 mg/kg (i.p.), inhibited antigen-induced bronchoconstriction and eosinophil accumulation in the bronchoalveolar lavage fluid (BALF) of guinea-pigs. These results suggest that NZ-107 has anti-allergic action including inhibition of eosinophil accumulation in an antigen-challenged airway lesion in rats and guinea-pigs. The anti-allergic action of this agent is thought to be due to its action as a histamine and LT antagonist and its consequent inhibition of antigen-induced histamine release.     

A novel cloverleaf structure found in mammalian mitochondrial tRNA(Ser) (UCN).

Bovine mitochondrial tRNA(Ser) (UCN) has been thought to have two U-U mismatches at the top of the acceptor stem, as inferred from its gene sequence. However, this unusual structure has not been confirmed at the RNA level. In the course of investigating the structure and function of mitochondrial tRNAs, we have isolated the bovine liver mitochondrial tRNA(Ser) (UCN) and determined its complete sequence including the modified nucleotides. Analysis of the 5'-terminal nucleotide and enzymatic determination of the whole sequence of tRNA(Ser) (UCN) revealed that the tRNA started from the third nucleotide of the putative tRNA(Ser) (UCN) gene, which had formerly been supposed. Enzymatic probing of tRNA(Ser) (UCN) suggests that the tRNA possesses an unusual cloverleaf structure with the following characteristics. (1) There exists only one nucleotide between the acceptor stem with 7 base pairs and the D stem with 4 base pairs. (2) The anticodon stem seems to consist of 6 base pairs. Since the same type of cloverleaf structure as above could be constructed only for mitochondrial tRNA(Ser) (UCN) genes of mammals such as human, rat and mouse, but not for those of non-mammals such as chicken and frog, this unusual secondary structure seems to be conserved only in mammalian mitochondria.     

SV40 T-antigen is required for maintenance of immortal growth in SV40-transformed human fibroblasts.

Two lines of immortal human fibroblasts were isolated following transfection of TIG-3 cells with plasmid DNA, pMT-1ODtsA, that contained SV40 early gene with a deletion in replication origin and ts mutation in coding sequence for T-antigen. These cells continued proliferation at 34 degrees C, over 565 population doubling level (PDL) which is far over the limited division potential of untransformed normal TIG-3 of 70-80 PDL. When the culture temperature was shifted to 40 degrees C after 70 PDL, they ceased proliferation immediately. One of these immortal clones, SVts8, lost its ts phenotype after retransformation with wtT-antigen gene. These results indicated that the function of intact T-antigen is required for maintenance of immortal proliferation, at least in one of the SV40 transformed immortal clones.     

A male-specific renal cytochrome P-450 isozyme(s) responsible for mutagenic activation of 3-methoxy-4-aminoazobenzene in mice

Renal microsomes from male mice (BALB/c, DBA/2 and BALB/c x DBA/2 F1) showed about 10-fold greater activity for mediating mutagenic activation of 3-methoxy-4-aminoazobenzene (3-MeO-AAB) toward Salmonella typhimurium TA98 than did the corresponding hepatic microsomes, as compared on the basis of nmol of microsomal cytochrome P-450. On the other hand, female renal microsomes and other extrahepatic microsomes (lung, small intestine and colon) in both sexes of mice showed little or no activity for converting 3-MeO-AAB to mutagen(s). The mutagenic activation of 3-MeO-AAB with the male renal enzyme(s) was definitely inhibited by cytochrome P-450 inhibitors, 7,8-benzoflavone and SKF 525A. All these findings suggest that in mice, there is a male-specific renal 3-MeO-AAB activation enzyme(s), a cytochrome P-450 isozyme(s), which is different, at least in proportion and/or in nature, from hepatic cytochrome P-450 isozymes.     

Changes in mRNA of Vigna mungo Cotyledons during Seed Germination

Quantitative and qualitative changes of mRNA in Vigna mungocotyledons during seed germination have been investigated. TotalRNA is higher in dry cotyledons and declines during germination.Poly(A)⁺ RNA also is present at a relatively high level in drycotyledons, increases slightly during the first day of germination,and then decreases. Polysomal RNA is very low in dry cotyledonsbut increases rapidly during the first day of germination, andthen declines. The translational activity of the mRNA in a wheatgerm cell-free system is low on day 0 but increases rapidlyon day 1 of germination. Two-dimensional gel electrophoresisof in vitro translation products reveals that many new peptidesare synthesized on day 1 of germination. Synthesis of most ofthese polypeptides continue throughout 5 days of germination. Change in the mRNA population during germination has been investigatedusing cDNA against poly(A)⁺ RNA from 3-day-old cotyledons. Withtotal RNA of day 3 and 5, the cDNA strongly hybridized withRNA similar in size to 25 S ribosomal RNA, but no specific bandsare detected with samples of day 0 or 1. With poly(A)⁺ RNA ofday 5 or 1, the cDNA tends to hybridize with RNAs of relativelysmall molecular size. Cordycepin and -amanitin prevent the increasein poly (A)⁺ RNA content and the appearance of new mRNAs duringthe first day of germination. ¹Present address: Division of Regulation of Macromolecular Function,Institute for Protein Research, Suita City, Osaka 565, Japan. (Received January 13, 1986; Accepted June 10, 1986)     

Phosphorylated tau protein is integrated into paired helical filaments in Alzheimer's disease

Antisera to paired helical filaments (PHF) were found to contain a significant amount of tau antibodies specific for a phosphorylated form, but only a negligible amount of those specific for a non-phosphorylated form. Also, the phosphorylated tau-specific antibodies, but not the non-phosphorylated tau-specific ones, labeled neurofibrillary tangles isolated in the presence of sodium dodecyl sulfate (SDS) and stained both tangles and senile plaque neuritis in fixed tissue sections in a very similar way to as the whole antiserum did. Taken together, these results strongly suggest that a major antigenic determinant of PHF is phosphorylated tau itself.     

Structures of sugar chains of human kidney gamma-glutamyltranspeptidase

gamma-Glutamyltranspeptidase purified from human kidneys contains 4-5 asparagine-linked sugar chains in each molecule. The sugar chains were released from the polypeptide portion of the enzyme by hydrazinolysis as oligosaccharides and separated by paper electrophoresis into one neutral and two acidic fractions. By sequential exoglycosidase digestion and methylation analysis, the neutral fraction, which comprised 69% of total oligosaccharides, was shown to be a mixture of bisected bi- and triantennary complex-type sugar chains with and without a fucose on the proximal N-acetylglucosamine residue and with Gal beta 1----4GlcNAc and/or Gal beta 1----4(Fuc alpha 1----3)GlcNAc groups in their outer chain moieties. The acidic oligosaccharide fractions were mixtures of mono- and disialyl derivatives of bisected triantennary complex-type oligosaccharides with Gal beta 1----4GlcNAc and/or Gal beta 1----4(Fuc alpha 1----3)GlcNAc group in their outer chain moieties. Some of the outer chains of the acidic oligosaccharides were considered to be sialylated X-antigenic structures.     

Specific interaction between tRNA and its cognate amino acid as detected by circular dichroism and fluorescence spectroscopy

Specific interaction was detected between tRNA and its cognate L-amino acid by circular dichroism (CD) and fluorescence spectroscopy; fluorescence spectral change with saturation was observed when tRNAAsp was titrated only with L-aspartic acid, but not with D-aspartic acid, L- and D-glutamic acids. It was also the case for tRNA2Glu and L-glutamic acid as detected by CD. These results are consistent with the hypothesis that the anticodon, or the complex of four nucleotides (C4N) of the anticodon three bases and the discriminator base in a tRNA (Shimizu, M., J. Mol. Evol. (1982) 18, 297-303) participates in recognition of amino acids.     

Suppressors of temperature-sensitive mutations in a ribosomal protein gene, rpsL (S12), of Escherichia coli K12

Summary Temperature-sensitive (ts) mutations were isolated within a ribosomal protein gene (rpsL) of Escherichia coli K12. Mutations were mapped by complementation using various transducing phages and plasmids carrying the rpsL gene, having either a normal or a defective promoter for the rpsL operon. One of these mutations, ts118, resulted in a mutant S12 protein which behaved differently from the wild-type S12 on CM-cellulose column chromatography. Suppressors of these ts mutations were isolated and characterized; one was found to be a mutation of a nonribosomal protein gene which was closely linked to the RNAase III gene on the E. coli chromosome. This suppressor, which was recessive to its wild-type allele, was cloned into a transducing phage and mapped finely. A series of cold-sensitive mutations, affecting the assembly of ribosomes at 20°C, was isolated within the purL to nadB region of the E. coli chromosome and one group, named rbaA, mapped at the same locus as the suppressor mutation, showing close linkage to the RNAase III gene.     

A spin-label study on human high density lipoprotein

Human plasma high density lipoproteins (HDL) have been labeled with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)maleimide (NEM-TEMPO). The spin-labeled HDL exhibited an ESR spectrum containing signals of both strongly immobilized and weakly immobilized components by the reaction with a high concentration of NEM-TEMPO, while an ESR spectrum containing only signals of a strongly immobilized component range between 4 degrees C and 37 degrees C, the signal height of the strongly immobilized component exhibited reversible temperature-dependent changes, whereas that of the weakly immobilized component changed irreversibly at temperatures above 25 degrees C. The activation energy of the irreversible change was estimated to be 26 kcal per mol. The strongly immobilized component was derived from NEM-TEMPO which modified apolipoprotein A-I covalently, while the weakly immobilized component was derived from NEM-TEMPO noncovalently bound to HDL. The rate of binding of NEM-TEMPO to either the strongly binding or weakly binding sites and the number of the strongly binding sites in apolipoprotein A-I were estimated to be 125 M-1.day-1 and 1.78, respectively. The binding of NEM-TEMPO to the strongly binding sites was suppressed greatly by pretreatment of HDL with 2,4,6-trinitrobenzene sulfonic acid (TNBS). The slow reaction and suppression with TNBS suggest that NEM-TEMPO binds to some amino acid residue, probably a lysine residue, in apoprotein A-I. The strongly immobilized and weakly immobilized components were reduced almost completely by ascorbate at the same rate, 0.048 min-1 at pH 7.4 and at 4 degrees C.