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)     

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.     

Intermediate epithelium lining the mouse auditory tube

In the newborn mouse, the auditory tube is lined throughout the pharyngeal orifice to the tympanic orifice with ciliated columnar epithelium. In the adult mouse, the tube is divided into membranous and cartilaginous parts. The membranous part is covered by the ciliated columnar epithelium, while the cartilaginous part by varying types of epithelium ranging from ciliated columnar to stratified squamous type. It is suggested that the varying types of epithelium correspond to the 'intermediate epithelium', and that ciliated columnar epithelium transforms in part to stratified squamous epithelium by passing through the intermediate epithelium.     

The Ca2+ influx induced by β-amyloid peptide 25–35 in cultured hippocampal neurons results from network excitation

Although a neurotoxic role has been postulated for the β-amyloid protein (βAP), which accumulates in brain tissues in Alzheimer's disease, a precise mechanism underlying this toxicity has not been identified. The peptide fragment consisting of amino acid residues 25 through 35 (βAP25-35), in particular, has been reported to be toxic in cultured neurons. We report that βAP25-35, applied to rat hippocampal neurons in culture, caused reversible and repeatable increases in the intracellular Ca²⁺ concentration ([Ca²⁺]_i), as measured by fura 2 fluorimetry. Furthermore, βAP25-35 induced bursts of excitatory potentials and action potential firing in individual neurons studied with whole cell current clamp recordings. The βAP25-35–induced [Ca²⁺]_i elevations and electrical activity were enhanced by removal of extracellular Mg²⁺, and they could be blocked by tetrodotoxin, by non-N-methyl-D -aspartate (NMDA) and NMDA glutamate receptor antagonists, and by the L-type Ca²⁺ channel antagonist nimodipine. Similar responses of bursts of action potentials and [Ca²⁺]_i increases were evoked by βAP1-40. Responses to βAP25-35 were not prevented by pretreatment with pertussis toxin. Excitatory responses and [Ca²⁺]_i elevations were not observed in cerebellar neuron cultures in which inhibitory synapses predominate. Although the effects of βAP25-35 depended on the activation of glutamatergic synapses, there was no enhancement of kainate- or NMDA-induced currents by βAP25-35 in voltage-clamp studies. We conclude that βAP25-35 enhances excitatory activity in glutamatergic synaptic networks, causing excitatory potentials and Ca²⁺ influx. This property may explain the toxicity of βAP25–35. © 1995 John Wiley & Sons, Inc.     

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.     

The effect of three novel thromboxane A2 receptor antagonists (S-1452, AA-2414 and ONO-3708) on the increase in pulmonary pressure caused by Forssman anaphylaxis in guinea-pigs.

The effects were studied of three novel thromboxane A2 (TXA2) receptor antagonists (S-1452, AA-2414 and ONO-3708) on the increase in pulmonary pressure caused by Forssman anaphylaxis in guinea-pigs. Three TXA2 antagonists at doses of between 1 and 10 mg/kg administered orally 1 h before the challenge clearly inhibited the pulmonary pressure increase. At a dose of 10 mg/kg, all three antagonists inhibited the pulmonary pressure increase caused by leukotriene D4 (LTD4) and U-46619, but not that caused by histamine. The decrease in peripheral platelet counts caused by Forssman anaphylaxis was also clearly inhibited by the three TXA2 antagonists. However, the decreased peripheral leukocyte counts were unaffected by the three agents. The decrease in serum complement activity (CH50) was inhibited by S-1452 and AA-2414 at a dose of 10 mg/kg. In bronchoalveolar lavage fluid (BALF), significant increases in eosinophils and neutrophils were observed after Forssman anaphylaxis. Three TXA2 antagonists at a dose of 10 mg/kg (except for AA-2414 on eosinophils) did not affect the changes of leukocyte counts in BALF. Moreover, increases in the TXB2 and 6-keto-PGF1 alpha levels of the BALF brought about by Forssman anaphylaxis were unaffected by the three TXA2 receptor antagonists. Histamine and LTD4 were not changed in the BALF after Forssman anaphylaxis. These results indicate the efficacy of TXA2 receptor antagonists on the increase in pulmonary pressure caused by Forssman anaphylaxis in guinea-pigs by direct antagonism to released TXA2.