Amino acid sequence of the nonsecretory ribonuclease of human urine

The amino acid sequence of a nonsecretory ribonuclease isolated from human urine was determined except for the identity of the residue at position 7. Sequence information indicates that the ribonucleases of human liver and spleen and an eosinophil-derived neurotoxin are identical or very closely related gene products. The sequence is identical at about 30% of the amino acid positions with those of all of the secreted mammalian ribonucleases for which information is available. Identical residues include active-site residues histidine-12, histidine-119, and lysine-41, other residues known to be important for substrate binding and catalytic activity, and all eight half-cystine residues common to these enzymes. Major differences include a deletion of six residues in the (so-called) S-peptide loop, insertions of two, and nine residues, respectively, in three other external loops of the molecule, and an addition of three residues at the amino terminus. The sequence shows the human nonsecretory ribonuclease to belong to the same ribonuclease superfamily as the mammalian secretory ribonucleases, turtle pancreatic ribonuclease, and human angiogenin. Sequence data suggest that a gene duplication occurred in an ancient vertebrate ancestor; one branch led to the nonsecretory ribonuclease, while the other branch led to a second duplication, with one line leading to the secretory ribonucleases (in mammals) and the second line leading to pancreatic ribonuclease in turtle and an angiogenic factor in mammals (human angiogenin). The nonsecretory ribonuclease has five short carbohydrate chains attached via asparagine residues at the surface of the molecule; these chains may have been shortened by exoglycosidase action.(ABSTRACT TRUNCATED AT 250 WORDS)     

In Vitro HIV-1 LTR Integration into T-Cell Activation Gene CD27 Segment and the Decoy Effect of Modified-Sequence DNA

Integration into the host genome is an essential step in the HIV-1 life cycle. However, the host genome sequence that is favored by HIV-1 during integration has never been documented. Here, we report that CD27, a T cell activation gene, includes a sequence that is a target for in vitro HIV-1 cDNA integration. This sequence has a high affinity for integrase, and the target nucleotides responsible for this higher affinity were identified using a crystal microbalance assay. In experiments involving a segment of the CD27 gene, integration converged in the target nucleotides and flanking sequence DNA, indicating that integration is probably dependent upon the secondary structure of the substrate DNA. Notably, decoy modified CD27 sequence DNAs in which the target nucleotides were replaced suppressed integration when accompanying the original CD27 sequence DNA. Our identified CD27 sequence DNA is useful for investigating the biochemistry of integrase and for in vitro assessment of integrase-binding inhibitors.     

Lack of association and linkage between HLA and familial polyposis coli

Summary We investigated possible association of and linkage between HLA and familial polyposis coli (FPC). In 182 individuals from 66 pedigrees of FPC and 108 individuals from a normal population, HLA-A,-B, and-C antigens were determined. When the frequencies of HLA antigens in 66 unrelated patients and in normal controls were compared, no association of FPC with HLA was observed. For the linkage analysis, HLA haplotypes of 17 affected sib pairs were investigated by the affected sib pair method. The number of pairs which shared two, one, and no haplotypes identical by descent was not significantly different from the number expected with random occurrence (P>0.95). Finally, seven families were analyzed using Morton's sequential test. A maximum lod score of-0.056 at a recombination fraction of 0.4, and a lod of-3.089 at a recombination fraction of 0.05 were obtained. Therefore, there is neither an association of nor linkage between FPC and HLA.     

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.     

Modification of a ribonuclease from Rhizopus sp. with 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide p-toluenesulfonate

The carboxyl group in a ribonuclease from Rhizopus sp. (RNase Rh) was modified by a water-soluble carbodiimide, 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide p-toluenesulfonate (CMC). From the relation between the extent of modification and the enzymatic activity, it was concluded that at least the modification of two carboxyl groups seemed to induce the loss in enzymatic activity. In the presence of 1 M cytidine, RNase Rh activity was protected from the CMC-modification. Under conditions in which the enzyme was inactivated to 20% activity, about 70% of the enzymatic activity was retained in the presence of cytidine. The inactivation of the RNase Rh pre-treated with CMC in the presence of cytidine with [14C]CMC indicated that the RNase Rh lost its enzymatic activity with the incorporation of about one [14C]CMC. Therefore, it could be concluded that one carboxyl group is involved in the active site of RNase Rh. The binding of the CMC-modified RNase Rh with 2'-AMP was studied spectrophotometrically. The affinity of the modified RNase Rh towards 2'-AMP decreased markedly upon CMC modification.     

Proteolytic Activity against Model Peptides of the Cell Wall Lytic Enzyme from Chlamydomonas

A cell wall lytic enzyme (gamete wall-autolysin) from Chlamydomonasreinhardtii specifically cleaved several synthetic model peptides,-neo-endorphin, dynorphin (1–13), neurotensin and mastoparan,at the peptide bonds between consecutive hydrophobic amino-acidresidues. The cleavage was not significantly affected by high-saltconditions which are known to inhibit digestion of the cellwall. (Received December 14, 1989; Accepted April 5, 1990)     

Qualitative and quantitative difference in mutation induction between carbon- and neon-ion beams in normal human cells.

We investigated the difference in cell-killing effect and mutation induction between carbon- and neon-ion beams in normal human cells. Carbon- and neon-ion beams were accelerated by the Riken Ring Cyclotron (RRC) at the Institute of Physical and Chemical Research in Japan. Cell-killing effect was measured as the reproductive cell death using the colony formation assay. Mutation induction at the HPRT locus was detected to measure 6-thioguanine-resistant clones. The mutation spectrum of the deletion pattern of exons of induced mutants was analyzed using the multiplex polymerase chain reaction (PCR). Cell-killing effect was almost the same between carbon- and neon-ion beams with similar linear energy transfer (LET) values, while there observed a large difference in mutation frequency. Furthermore, in the case of neon-ion beams 60% of mutants showed total deletions and 35-40% showed partial deletions, while 95-100% of carbon-ion induced mutants showed total deletions. The results suggest that different ion species may cause qualitative and quantitative difference in mutation induction even if the LET values are similar.     

Identification of lactotropes (PRL cells), somatotropes (GH cells), corticotropes (ACTH cells) and thyrotropes (TSH cells) in the pituitary gland of the musk shrew, Suncus murinus L. (Insectivora), by immunohistochemistry

By means of immunohistochemistry, lactotropes, somatotropes, corticotropes and thyrotropes in the pituitary of the male musk shrew, Suncus murinus L., were identified at the electron-microscopic level. Lactotropes were classified into three types: type I containing large (200-450 nm in diameter) round secretory granules, type II with medium-sized (150-250 nm in diameter) ones and type III with small (50-150 nm in diameter) ones. Somatotropes were also classified into type I somatotropes that contain large (450 nm in diameter) spherical secretory granules and type II somatotropes containing comparatively small (300 nm in diameter) round granules. Both type I and II somatotropes were small and sometimes contained rod-shaped granules. Corticotropes were round or oval cells with round secretory granules in various densities and sizes (150-500 nm in diameter) scattered all over the cytoplasm. Thyrotropes were angular or polyhedral cells containing electron-transparent round secretory granules (200-300 nm in diameter) and large irregularly shaped granules with a maximum diameter of about 1,500 nm. Each type of the cells may be distinguished by its respective ultrastructural characteristics alone without the aid of immunohistochemistry.     

A mutant Escherichia coli tyrosyl-tRNA synthetase utilizes the unnatural amino acid azatyrosine more efficiently than tyrosine

Alloproteins, proteins that contain unnatural amino acids, have immense potential in biotechnology and medicine. Although various approaches for alloprotein production exist, there is no satisfactory method to produce large quantities of alloproteins containing unnatural amino acids in specific positions. The tyrosine analogue azatyrosine, l-beta-(5-hydroxy-2-pyridyl)-alanine, can convert the ras-transformed phenotype to normal phenotype, presumably by its incorporation into cellular proteins. This provided the stimulus for isolation of a mutant tyrosyl-tRNA synthetase (TyrRS) capable of charging azatyrosine to tRNA. A plasmid library of randomly mutated Escherichia coli tyrS (encoding TyrRS) was made by polymerase chain reaction techniques. The desired TyrRS mutants were selected by screening for in vivo azatyrosine incorporation of E. coli cells transformed with the mutant tyrS plasmids. One of the clones thus isolated, R-6-A-7, showed a 17-fold higher in vivo activity for azatyrosine incorporation than wild-type TyrRS. The mutant tyrS gene contained a single point mutation resulting in replacement of phenylalanine by serine at position 130 in the protein. Structural modeling revealed that position 130 is located close to Asp(182), which directly interacts with tyrosyladenylate. Kinetic analysis of aminoacyl-tRNA formation by the wild-type and mutated F130S TyrRS enzymes showed that the specificity for azatyrosine, measured by the ratios of k(cat)/K(m) for tyrosine and the analogue, increased from 17 to 36 as a result of the F130S mutation. Thus, the high discrimination against azatyrosine is significantly reduced in the mutant enzyme. These results suggest that utilization of F130S TyrRS for in vivo protein biosynthesis may lead to efficient production of azatyrosine-containing alloproteins.     

Different binding sites for the neuropeptide Y Y1 antagonists 1229U91 and J-104870 on human Y1 receptors

The peptidic Y1 antagonist 1229U91 and the non-peptidic antagonist J-104870 have high binding affinities for the human Y1 receptor. These Y1 antagonists show anorexigenic effects on NPY-induced feeding in rats, although they have completely different structures and molecular sizes. To identify the binding sites of these ligands, we substituted amino acid residues of the human Y1 receptor with alanine and examined the abilities of the mutant receptors to bind the radio-labeled ligands. Alanine substitutions, F98A, D104A, T125A, D200A, D205A, L215A, Q219A, L279A, F282A, F286A, W288A and H298A, in the human Y1 receptor lost their affinity for the peptide agonist PYY, but not for 1229U91 and J-104870, while L303A and F173A lost affinity for 1229U91 and J-104870, respectively. N283A retained its affinity for 1229U91, but not for PYY and J-104870. Y47A and N299A retained their affinity for J-104870, but not for PYY and 1229U91. W163A and D287A showed no affinity for any of the three ligands. Taken together, these data indicate that the binding sites of 1229U91 are widely located in the shallow region of the transmembrane (TM) domain of the receptor, especially TM1, TM6 and TM7. In contrast, J-104870 recognized the pocket formed by TM4, TM5 and TM6, based on the molecular modeling of the Y1 receptor and J-104870 complex. In conclusion, 1229U91 and J-104870 have high affinities for Y1 receptors using basically different binding sites. D287 of the common binding site in the TM6 domain could be crucial for the binding of Y1 antagonists.