Minoru Nomoto

Complete amino acid sequence of boar protamine

Abstract The complete amino acid sequence of boar protamine was determined. In order to specify cleaving points for enzymatic fragmentation, two S-alkyl derivatives of the protamine were used. 1. The sequences other than arginine stretches were determined by analysis of tryptic peptides from the S-carboxymethyl-protamine. The sequence of the central arginine-clustered region was determined by stepwise degradation from the C-terminus of the thermolysin core-fragment with carboxypeptidase A and B, and then with acid carboxy-peptidase. 2. S-Methylation of cysteine residues of the protamine was found to introduce a new point for thermolysin and chymotryptic cleavage. Thus, thermolysin cleavage of the chymotryptic core-fragment resulted in oligo-arginine peptides with methylcysteine at the N-terminus, by which the sequence of arginine-clusters was confirmed. 3. The complete sequence was deduced by overlap of these sequenced peptides, which is: Ala-Arg-Tyr-Arg-Cys2-Arg-Ser-His-Ser-Arg-Ser-Arg-Cys-Arg-Pro-Arg4-Cys-Arg6-Cys2-Pro-Arg5-Ala-Val-Cys2- Arg2-Tyr-Thr-Val-Ile-Arg-Cys-Arg2Cys. Boar protamine, as well as bull protamine, is composed of the less basic amino- and carboxy-terminal domains and the central arginine-clustered domain, and 80% homology was found between boar and bull protamines.

Synapse formation by autonomic nerves in the previously denervated neuromuscular junctions of the feline intrinsic laryngeal muscles

Nerve terminals of unknown origin at the previously denervated neuromuscular junctions (NMJ) of the feline intrinsic laryngeal muscles were investigated. Until 3 weeks after the transection of the recurrent laryngeal nerve (RLN), no axons were observed in the Büngners bands and the NMJ, accompanied by a marked decrease of autonomic nerves around the blood vessels. At 5-6 weeks nerve varicosities labeled by 5-hydroxydopamine (5-OHDA) were observed in the Büngners bands together with an increase of autonomic nerves around the blood vessels. At 9-30 weeks (8 cases) nerve terminals were found at the NMJ in all cases. Even if the ipsilateral vagosympathetic trunk was transected at 17 weeks, nerve terminals were found at all the NMJ 3 weeks after this treatment, indicating that nerve terminals were not from the original RLN. These nerve terminals were considered to be autonomic because nerve terminals labeled by 5-OHDA were observed. Furthermore, in the case of the removal of the superior cervical ganglion (SCG) and the nodose ganglion at 21 weeks, though nerve varicosities were found in the Büngners bands, nerve terminals were not found 9 days after this treatment, suggesting that the ipsilateral SCG possibly played an important role. In electromyographic findings fibrillation-like activities were recognized in 8/11 cases after 5 weeks. The relationship of this phenomenon to fibrillation and muscle atrophy was discussed.

Misdirected reinnervation in the feline intrinsic laryngeal muscles after long-term denervation

Nerve terminals which resembled the autonomic nerve varicosities were found at all neuromuscular junctions of the feline intrinsic laryngeal muscles after 21 months of unilateral laryngeal paralysis. The structure of muscle fibers was preserved throughout this period, although there was no evidence of regeneration of transected recurrent laryngeal nerve. In the cricothyroid muscle, misdirected reinnervation was recognized in the different pathways from the other intrinsic laryngeal muscles.

Isolation and characterization of nuclear basic protein (protamine) from boar spermatozoa

1. Sperm nuclei were isolated and purified from boar semen by a procedure involving differential solubilization of sperm tail and acellular materials by brief exposure to reducing reagent in the presence of cationic detergent, and sedimentation through 60% sucrose. The weight ratio of DNA:RNA: total protein: protamine in this preparation was 1.00: 0.02: 1.05: 0.75, and the molar ratio of phosphorus to arginine was 1.12. 2. Boar protamine was extracted with cold acid from ethanol precipitate of reduced and carboxymethylated nuclei in 6 M guanidine hydrochloride and purified by ion-exchange chromatography on CM-cellulose. The molecular weight of the protamine was estimated to be 6600 by the gel filtration method. The protamine consisted of a single amino terminus alanine and either half-cystine or arginine as carboxy terminus, and was composed of Thr, Ser3, Pro2, Ala2, Val2, Ile, His, Half-cystine9-10 and Arg26 . 3. Chymotryptic digestion gave rise to a single amino-terminal peptide, Ala-Arg-Tyr, and two carboxy-terminal peptides, Thr-Val-Ile-Arg-Cys-Arg2-Cys and Thr-Val-Ile-Arg-Cys-Arg2, which confirmed the heterogeneity of the protamine at the carboxy-terminal end.

Role of capsaicin-sensitive trigeminal nerves in development of hyperreactive nasal symptoms in guinea pig model of nasal allergy.

The effect of capsaicin pretreatment on frequency of sneezing, decrease of nasal patency, and increase of vascular dye leakage induced by antigen or histamine challenge on the guinea pig nasal mucosa was investigated. The animals were sensitized intraperitoneally with ovalbumin. Capsaicin pretreatment significantly inhibited sneezing induced by nasal challenge with histamine and antigen, indicating that capsaicin-sensitive sensory nerves constitute an afferent pathway of the sneezing reflex in nasal allergy. Although capsaicin pretreatment tended to inhibit the decrease of nasal patency and the increase of vascular dye leakage of the nasal mucosa induced by antigen challenge, this tendency was not statistically significant. The present study indicated that the participation of a local reflex via capsaicin-sensitive trigeminal nerves in nasal vascular responses observed after antigen challenge in the guinea pig model of nasal allergy is rather small compared to the large direct vascular effects of chemical mediators released from basophilic cells in the nasal mucosa.

Synovial osteochondromatosis of the temporo-mandibular joint

A rare case of synovial osteochondromatosis of the temporo-mandibular joint is presented. Important diagnostic information can be obtained by CT, MRI, 99m Tc bone scan and aspiration biopsy. Observation using a light microscope showed mild cellular atypia, but the hallmarks characteristic of chondrosarcoma were not found. Observation using an electron microscope showed the mature chondrocytes contained a well-developed rough endoplasmic reticulum. Histological evaluation indicated that the present case was benign synovial osteochondromatosis in an early stage.

Ultrastructural and Histochemical Study of the Neuromuscular Junctions in the Denervated Intrinsic Laryngeal Muscle of the Cat

Morphological changes and acetylcholinesterase (AchE) activity in the neuromuscular junctions (NMJ) of the normal and denervated posterior cricoarytenoid muscle (PCA muscle) of the cat were studied. Two days after denervation, the nerve terminals at the NMJ had almost disappeared. Six weeks after denervation, intensity of AchE activity at the former junctional site (FJS) was unchanged histochemically. At this stage, primary synaptic clefts were distorted and the Schwanns cells covered the FJS. Fourteen weeks after denervation, AchE activity at the FJS had decreased in contrast to that of the non-affected side. Calcitonin gene-related peptide (CGRP) was also investigated at the NMJ of the normal PCA muscles immunocytochemically. The present study shows that CGRP coexists with Ach in the nerve terminals of the PCA muscles and may be involved in the regulation of the contractile function of the intrinsic laryngeal muscle.

Effect of Diesel Exhaust on Guinea Pig Nasal Mucosa

In this study using guinea pigs, we investigated the effects of diesel exhaust (DE) containing diesel exhaust particulate (DEP) on 1) vascular permeability induced by histamine, 2) nasal mucosal permeability to horseradish peroxidase (HRP), and 3) eosinophilic epithelial infiltration. The vascular permeability induced by histamine was enhanced significantly and dose-dependently in DE-exposed guinea pigs. The HRP reaction products in epithelial cells and intercellular spaces were significantly and dose-dependently increased in those guinea pigs. Eosinophil infiltration into the epithelial layer was significantly increased in guinea pigs exposed to DE containing 3.2 mg/m3 DEP, and the reactivity of the nasal mucosa to histamine solution applied on the nasal mucosa was significantly enhanced in those guinea pigs. These findings suggest that DE may play an important role not only in promoting nasal hyperreactivity induced by the enhancement of absorption of antigen through the nasal epithelium, but also in inducing eosinophil infiltration in nasal mucosa and enhancing nasal mucosal reactivity.

Effect of psychic stimulation on plasma catecholamine concentrations and nasal patency in patients with nasal allergy

To compare the degree of sympathoadrenal and nasal vascular response to psychic stimulation between patients with nasal allergy and normal controls, we measured the changes in plasma norepinephrine and epinephrine concentrations and nasal patency elicited by cannulation into the forearm vein and mental arithmetic in 28 patients with nasal allergy and age- and sex-matched normal controls. Ten of the 28 allergy patients had markedly swollen, pale, edematous nasal mucosa and served as a subgroup of nasal allergy patients. Plasma catecholamine levels increased significantly, with a synchronous increase of nasal patency, during stimulation. Among the three groups, no statistical differences were observed in plasma catecholamine levels either at rest or during stimulation. There was no significant difference in extent of increase of nasal patency induced by stimulation in the total group of subjects with nasal allergy compared with normal controls. However, it was significantly smaller in a subgroup of nasal allergy patients having markedly pale, edematous swelling of the nasal mucosa.

Cytochemical Demonstration of Actin Filaments in Myoepithelial Cells of the Human Parotid Gland

Ultrastructurally, myoepithelial cells were shown to contain numerous fine filaments in their cytoplasm and resembled smooth muscle cells. The myoepithelial cell of the salivary gland has been considered to play an important role in the secretion of saliva. The present study showed that all the thin filaments (actin filaments) in the myoepithelial cell of the human parotid gland bound heavy meromyosin (HMM) and formed characteristic arrowhead structures. These filaments ran in two opposite directions with the poles at different ends. On the other hand, there was no binding of HMM with thicker filaments (10-nm filaments), plasma membrane, nuclear membrane, collagen fibrils, basement membrane or other cytoplasmic organelles. The present results strongly suggest that myoepithelial cells possess a contractile function parallel to the long axis of the cell for supporting the secretion of saliva in the parotid gland.