Studies on the structure and function of subtilisin E by protein engineering.
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Amino Acid Sequence↗
Biomedical subjects
M Yamasaki
Publications and source records attributed to M Yamasaki.
Protein transport in the permeabilized cell of Schizosaccharomyces pombe.
We reconstituted a protein translocation-transport system composed of permeabilized spheroplasts (P-cells) of the fission yeast Schizosaccharomyces pombe and the precursor of alpha sex pheromone, prepro-alpha-factor of the budding yeast Saccharomyces cerevisiae. We found that P-cells prepared from the spheroplasts formed in 0.7M KCl as an osmotic stabilizer had the activity to transport pro-alpha-factor to the Golgi apparatus. Electron microscopic observations showed that membranes were preserved more intact in the P-cells prepared from the spheroplasts formed in 0.7M KCl than in 0.7M sorbitol. A glycoprotein of S. pombe contains galactose residues, and we detected incorporation of radiolabeled galactose residues into the anti-prepro-alpha-factor immunoprecipitable fractions in this S. pombe system, but not in the S. cerevisiae system. This paper reports that a heterologous system of in vitro protein transport was performed, and prepro-alpha-factor has the signals necessary for early steps of the transport in S. pombe.
Biological Transport↗
The reflex bradycardia evoked by brain ischemia and its relation to aortic A- and C-fiber baroreceptors.
In urethane-anesthetized rabbits with the right aortic nerve (RAN) sectioned, we examined the reflex heart rate (HR) responses during brain ischemia for approximately 30 s, by applying the anodal block to the unsectioned left aortic nerve (LAN) and, subsequently, by denervating the LAN. The maximum decreases in HR occurred at around 30 s after the onset of brain ischemia. The anodal block used in this study selectively inhibited the aortic A-fiber conduction but did not inhibit the volley of aortic C-fibers. The maximum HR fall responses to brain ischemia with and without the anodal block were 143 +/- 7 and 183 +/- 7 beats/min, respectively. When the maximum value of HR fall during brain ischemia in the absence of aortic nerve signals was subtracted from these two values, the reflex HR fall responses to brain ischemia with aortic C and A baroreceptor activation were 98 +/- 7 and 43 +/- 8 beats/min, respectively. In another series of experiments used for the same techniques, the reflex fall in HR seen during brain ischemia with the anodal block was totally abolished by vagotomy. The results indicate that the ability of aortic C baroreceptors becomes more prominent on the magnitude of brain ischemia-induced reflex bradycardia as compared to that of aortic A baroreceptors.
Action Potentials↗
Serodiagnosis of hepatitis C virus infection by ELISA for antibodies against the putative core protein (p20c) expressed in Escherichia coli.
The putative core gene of hepatitis C virus (HCV) was incorporated into a plasmid vector (pCC5-J4), and expressed in Escherichia coli. The product of 180 amino acids (p20c) was purified by gel electrophoresis in the presence of sodium dodecyl sulfate, and used in enzyme-linked immunosorbent assay for antibodies against the putative core protein of HCV (anti-p20c). Anti-p20c was detected in 13 (1.5%) of 873 apparently healthy blood donors. It was detected in 205 (86.5%) of 237 patients with acute or chronic non-A, non-B (NANB) hepatic disease, significantly more frequently (p less than 0.01) than antibodies against the C100-3 protein encoded by nonstructural regions of HCV (anti-C100-3) that was found in 178 (75.1%). Anti-p20c developed in the circulation of a patient with acute NANB hepatitis much earlier than anti-C100-3. HCV RNA was detected by polymerase chain reaction in serum samples from blood donors positive for anti-p20c in high titers, one of which was negative for anti-C100-3. These results indicated that anti-p20c would be useful in complementing anti-C100-3 for the diagnosis of NANB hepatitis and further decreasing the incidence of posttransfusion NANB hepatitis.
Adolescent↗
Immunohistochemical demonstration of elevated expression of epidermal growth factor receptor in the neoplastic changes of cervical squamous epithelium.
To evaluate epidermal growth factor (EGF) receptor expression in the neoplastic process of squamous cell epithelium of the uterine cervix, normal, premalignant, and malignant cervical tissues were examined for the presence of EGF receptor by the avidin-biotin immunoperoxidase techniques with a monoclonal antibody to EGF receptor. Although normal cervical epithelium did not show appreciable staining for EGF receptor, predominant staining for the receptor was observed in most dysplastic epithelia and carcinomas in situ. In invasive squamous carcinoma, there was a great difference in the immunohistochemically detected levels of EGF receptor among the histologic cell types. Large cell nonkeratinizing carcinoma and its keratinizing counterpart contained high levels of EGF receptor; small cell nonkeratinizing carcinoma lacked immunostainable EGF receptor. These results suggest that the elevated expression of EGF receptor may be involved in the initial stage of tumorigenesis of cervical squamous epithelium and that EGF receptor expression may be related to the differentiation or dedifferentiation of cervical squamous carcinoma cells.
Antibodies, Monoclonal↗
Sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for (anti-human T-cell leukemia virus type I) IgG in serum using recombinant gag p24(14-214) as antigen.
A sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for (anti-human T-cell leukemia virus type I) IgG (anti-HTLV-I IgG) in serum using recombinant gag p24(14-214) of HTLV-I is described. The recombinant gag p24(14-214) is soluble in the absence of detergents and allows the use of enzymes other than horseradish peroxidase as a label in the assays. The usefulness of recombinant gag p24(14-214) was examined with 305 sera characterized by other methods including gelatin particle agglutination, enzyme-linked immunosorbent assay (ELISA) using HTLV-I, and Western blotting. This assay was more sensitive than other methods using HTLV-I as antigen. The specificity could be tested by preincubation of test serum with excess of the recombinant protein. Most of negative and positive sera were discriminated. However, some results appeared to be false-positive or false-negative, and recombinant gag p24(14-214) was suggested to be useful, when used with other recombinant proteins and/or peptides, for improving the reliability of serodiagnosis by separately demonstrating antibodies against as many different epitopes of HTLV-I as possible. Anti-HTLV-I IgG in test serum, which had been incubated with excess of inactive beta-D-galactosidase to eliminate interference by anti-beta-D-galactosidase antibodies, was reacted simultaneously with 2,4-dinitrophenyl-bovine serum albumin-recombinant gag p24(14-214) conjugate and recombinant gag p24(14-214)-beta-D-galactosidase conjugate. The complex formed consisting of the three components was trapped onto polystyrene balls coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG.(ABSTRACT TRUNCATED AT 250 WORDS)
Evaluation Studies as Topic↗
Prominent inclusion effect of dimethyl-beta-cyclodextrin on photoisomerization of the thromboxane synthetase inhibitor (E)-4-(1-imidazoylmethyl)cinnamic acid.
The direct photoisomerization of (E)-4-(1-imidazoylmethyl)-cinnamic acid (IMC), a thromboxane synthetase inhibitor, to its (Z)-isomer at pH 2.0 was decelerated by beta-cyclodextrin (beta-CyD) and heptakis(2,6-di-O-methyl)-beta-cyclodextrin (DM-beta-CyD). The photostationary composition [(Z)-isomer:IMC ratio] was shifted in favor of IMC. These effects were much greater with DM-beta-CyD than with the parent beta-CyD. The quantum yield of the photoisomerization was significantly decreased by complex formation with beta-CyDs, whereas the extinction coefficient of the guest was only slightly decreased. This situation was in sharp contrast to those observed in less polar solvents and suggests that the suppressing mechanism with beta-CyD is different from that with less polar solvent systems. Spectroscopic studies (ultraviolet, circular dichroism, and nuclear magnetic resonance) indicated that IMC is tightly included in an axial mode in the cavity of DM-beta-CyD and that the rotation of the photoreactive site is sterically hindered. The results suggest that the suppressing effect of beta-CyDs on the photoisomerization of IMC results mainly from a steric origin.
Cyclodextrins↗
Neuroendocrine carcinoma of the uterus.
Neuroendocrine carcinomas of the cervix and endometrium were reviewed. They have been variously designated as carcinoid, argyrophil cell carcinoma, apudoma, small cell carcinoma, oat cell carcinoma, endocrine carcinoma, and neuroendocrine carcinoma, the last-mentioned term being preferred in this chapter. Adenocarcinomas with neuroendocrine cells are occasionally encountered in the cervix and endometrium. It is generally questioned whether they should be included in the spectrum of neuroendocrine carcinomas, although differential diagnosis between some such tumors of the gastrointestinal tract and neuroendocrine carcinoma is reported to be difficult. Since the majority of neuroendocrine carcinomas of the cervix are highly aggressive, it is important to establish the neuroendocrine nature in the cervical carcinomas. In addition to the characteristic histologic features and argyrophil stainability, immunohistochemical demonstration of several neuroendocrine markers may be helpful in diagnosing neuroendocrine carcinoma of the cervix. Ultrastructural demonstration of neurosecretory granules is almost decisive in establishing the tumor's neuroendocrine nature, but it is not applicable in all cases. Neuroendocrine carcinomas of the cervix have been treated by surgery, radiation therapy, and chemotherapy, but optimal treatment methods have not yet been established because of the rarity of the tumor. Finally, we have described a typical neuroendocrine carcinoma of the cervix and reported some data regarding its experimental study.
Adenocarcinoma↗
Myelination of two axons by a single Schwann cell.
A Schwann cell can form only one internode of myelin around an axon. However, we observed the formation by a single Schwann cell of myelin around two axons of different diameters in the sural nerve of a 45-year-old man with mononeuritis multiplex. Schwann cell processes spiraled in the same direction around each axon, forming mesaxons. The findings in this case appear to be an undescribed type of aberrant myelination.
Axons↗
ATPase activity of SopA, a protein essential for active partitioning of F plasmid.
The sopA, B, C genes of the F plasmid play an essential role in plasmid partitioning during cell division in Escherichia coli. In this paper, the products of the sopA and sopB genes were isolated and their biochemical activities studied. [alpha-32P]ATP was cross-linked to the SopA protein by UV irradiation; this cross-linking was observed only in the presence of magnesium ion, and was competitively inhibited in the presence of non-radioactive ATP, ADP and dATP, but not other NTPs or dNTPs. In contrast, no ATP binding activity was detected for the SopB protein. The SopA protein showed a modest magnesium ion-dependent ATPase activity and this activity was stimulated in the presence of DNA. The ATPase activity in the presence of DNA was further stimulated by addition of the SopB protein. However, the SopB protein alone failed to stimulate the ATPase activity.
Adenosine Triphosphatases↗
Application of fuzzy logic to the Apgar scoring system.
Apgar fuzzy expert system (AFES) applied fuzzy logic to the Apgar Scoring System (APG). We prepared the AFESs for both an inexperienced obstetrician (resident A) and an experienced obstetrician (specialist B) separately. We then compared their evaluations of the same 80 neonates at 1 min after birth using both the AFES and the APG. Analysis of the relationship of the general evaluation by the APG (scores below 6 or over 7) and the AFES (bad or good) to the umbilical arterial blood gases (pH below or above 7.20) showed that the sensitivities were 0.36 for the APG evaluation by resident A, 0.50 for the APG evaluation by specialist B, 0.60 for the AFES rating by resident A, and 0.71 for the AFES rating by specialist B. The specificities were 0.94, 0.95, 0.97 and 0.97, respectively. These results demonstrated that the AFES was capable of reflecting the recognition of the examiner.
Apgar Score↗
Analysis of adenoid cystic carcinoma treated by radiotherapy.
The records of 41 patients with adenoid cystic carcinoma of the head and neck region who had been treated with radiotherapy were reviewed. Local control was achieved in 72.3% in the cases with primary lesions at 5 years. The prognosis for tumors that arose in the major salivary glands was better than that for tumors that arose in the minor salivary glands; however, the difference was not statistically significant. In the minor salivary glands, early-stage tumors were well controlled with the use of radiation therapy alone. In spite of the high local control rate, the disease-free survival rate of the patients at 10 years was only 20.8%. Lung metastasis determined the prognosis.
Brachytherapy↗
Pulmonary C-fibers elicit both apneusis and tachypnea in the rabbit.
The effects of phenylbiguanide (PBG) on phrenic nerve and pulmonary C-fibers were studied in anesthetized spontaneously breathing rabbits with unilateral vagotomy. Right atrial injections of PBG at low (10 micrograms/kg) and high (100 micrograms/kg) dose resulted in a shallow tachypnea and apneusis followed by tachypnea, respectively, and these effects were blocked by procaine treatment of the vagus nerve. Also, the injection of PBG (100 micrograms/kg) still evoked the rapid shallow breathing preceded by apneusis in carotid chemoreceptor-denervated animals. Vigorous stimulation of pulmonary C-fibers by PBG (100 micrograms/kg) coincided with apneusis and the response was followed by a more modest increase in activity associated with tachypnea. Administration of PBG (10 micrograms/kg) into the right atrium caused an increase in pulmonary C-fiber activity associated with tachypnea. However, a small dose of PBG injected into the aortic circulation had no effect on the C-fiber activity but did inhibit respiration. These results suggest that the stimulation of pulmonary C-fibers via PBG injection can produce both inspiratory apnea and tachypnea.
Administration, Topical↗
Cholinergic and H1-receptor influences of histamine on slowly adapting pulmonary stretch receptor activity in the rabbit.
Afferent impulses of slowly adapting pulmonary stretch receptors (SARs) were obtained by dissecting fine slips from the left vagus nerve (LVN) and by leaving the rest of the nerve intact. In the same SAR preparation, changes of the receptor activity in response to right atrial injections of histamine (10 and 60 micrograms/kg) were successively examined before and after atropine (1 mg/kg), partial vagal efferent ablation, and mequitazine (1 mg/kg) in 10 rabbits. Administration of histamine led to an increase in the SAR activity, and this effect became more pronounced by increasing the dose of histamine. Atropine treatment diminished the responses of SARs to histamine at different doses. Partial vagal efferent ablation produced by denervation of the rest of the intact LVN slightly reduced the response of SARs to histamine at 10 micrograms/kg but had no significant effect on the SAR response to 60 micrograms/kg histamine. In the absence of vagal afferent and efferent activities on the left side, mequitazine, a potent H1-receptor blocker, completely blocked low- and high-dose effects of histamine on SARs. We compared the responses of the receptor activity to aerosol histamine (1 and 4%) and to topical application of histamine (0.1 ml, 0.025% and 0.1%) in six SAR preparations. The magnitude and duration of increased SAR activity became more prominent by increasing the concentration of histamine. The firing pattern and discharge rate of SARs following aerosol or intratracheal administration of histamine were similar to those after intra-atrial histamine. In addition, we also examined the excitatory responses of SAR activity to right atrial injections of histamine at 10 and 60 micrograms/kg before and after topical administration of atropine (0.1 ml, 1%, n = 6) or mequitazine (0.1 ml, 1%, n = 6) in 12 SAR preparations. Intratracheal atropine diminished the response of SARs to 10 micrograms/kg of histamine but had no significant effect on the response of SARs to histamine at 60 micrograms/kg. All the responses of SARs to histamine were completely blocked by topical application of mequitazine. These results suggest that the change of SAR activity produced by histamine at 10 micrograms/kg occurs mainly as a result of the release of acethylcholine (ACh) via the vagovagal reflex and that the activation of H1-receptors of the airway smooth muscle contributes importantly to the response of SARs to histamine at 60 micrograms/kg.
Acetylcholine↗
The relationship between clinical symptoms and anaerobic bacteria from infected root canals.
The purpose of this study was to investigate the correlation between the composition of bacterial flora from infected root canals and clinical symptoms. The materials evaluated consisted of 28 teeth from 25 patients with apical periodontitis. Eubacterium were found to be significantly related to acute or chronic clinical symptoms and Peptococcus, Peptostreptococcus, and Porphyromonas gingivalis to subacute clinical symptoms. We suggested that Peptococcus, Peptostreptococcus, Eubacterium, Porphyromonas, and Bacteroides were significantly related to percussion pain; Porphyromonas and Bacteroides were significantly related to odor in the infected root canals. Many Bacteroides were isolated from most of the infected root canals.
Adult↗
Endotoxin and gram-negative bacteria in the rat periapical lesions.
The purpose of this study was to measure the amount of endotoxin as well as to identify Gram-negative bacteria in experimental periapical lesions in rats. Molar pulps were exposed and infected and the amount of endotoxin in the periapical tissue of the right mandibular first molar was measured by Endospecy, while the colony number of Gram-negative bacteria was determined in the same region of the left mandibular first molar. In the control animals, the amount of endotoxin in the periapical tissues did not change at all during the experimental period, and no Gram-negative bacteria were isolated. In the experimental animals, the amount of endotoxin in the periapical tissues increased gradually from 1 to 70 days, and its level was significantly greater than that of control animals after 7 days. Gram-negative bacteria were isolated from the periapical tissues and their number gradually increased from 1 to 14 days (26 to 82%), but decreased at 21 days. It was approximately 60% from 28 to 70 days. The results of this study showed that the amount of endotoxin in the periapical tissues gradually increased with increasing time and that Gram-negative bacteria were isolated from the same region but did not increase in number concurrently with the increase in the amount of endotoxin.
Animals↗
Differential scanning calorimetric studies on bovine serum albumin: III. Effect of sodium dodecyl sulphate.
Measurements of differential scanning calorimetry (d.s.c.) have been made on the complex bovine serum albumin (BSA)--sodium dodecyl sulphate (SDS) under various conditions. There are two peaks P1 and P2 in the d.s.c. curve for BSA at pH 7 and in the absence of NaCl, indicating the presence of the heat-induced transition of BSA. There are three peaks P1, P2 and P3 in the curve for the system with the molar mixing ratio SDS/BSA = 1. With the increase in the amount of SDS, the peak P3 grows at the expense of P1 and P2. There is only a single peak P3 in the curve for the systems SDS/BSA > 7, and no peak at SDS/BSA = 50 and 100. There is a single peak P12 in the curve for BSA at pH 7 and in the presence of 0.05 M NaCl, indicating that the heat-induced transition is suppressed. There are two peaks P12 and P3 for the systems SDS/BSA = 1-5; the area ratio of the peak P3 to P12 increases with the increase in the amount of SDS. There is only a single peak P3 when SDS/BSA > 7, and no peak at SDS/BSA = 50. It is concluded that the peak P3 is a product of SDS regardless of the presence or absence of NaCl. Values of thermal denaturation temperature (Td) and enthalpy (delta H) of thermal denaturation indicate that the complex AD12 (A = BSA, D = SDS) is in the most thermostabilized state.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
The effect of amino acid deletion in subtilisin E, based on structural comparison with a microbial alkaline elastase, on its substrate specificity and catalysis.
Subtilisin from a wide variety of Bacillus species has been extensively investigated as a promising target for protein engineering. In this study, we analyzed the substrate specificity of B. subtilis subtilisin E based on the structure of a new alkaline elastase produced by the alkalophilic Bacillus strain Ya-B, which has very high elastolytic activity. Despite the high homology of the primary sequences of both enzymes (54% identical), alkaline elastase was found to lack four consecutive amino acids which, in subtilisin, have been shown by X-ray analysis to lie close to the P1 binding cleft. To examine the influence of such a deletion in subtilisin on its substrate specificity, we constructed several mutants missing four amino acids by site-directed mutagenesis. When assayed with synthetic peptides, elastin and casein as substrates, a mutant lacking Ser161-Thr162-Ser163-Thr164 showed considerably lower specific activity toward the substrates for subtilisin, and its substrate specificity approached that of alkaline elastase. The results indicate that the deletion in subtilisin E influences the catalytic efficiency as well as the P1 specificity, and that this region is, in part, responsible for the difference in specificity between the two enzymes.
Amino Acid Sequence↗