Crystal structure of N-carbamyl-D-amino acid amidohydrolase with a novel catalytic framework common to amidohydrolases.

BACKGROUND: N-carbamyl-D-amino acid amidohydrolase (DCase) catalyzes the hydrolysis of N-carbamyl-D-amino acids to the corresponding D-amino acids, which are useful intermediates in the preparation of beta-lactam antibiotics. To understand the catalytic mechanism of N-carbamyl-D-amino acid hydrolysis, the substrate specificity and thermostability of the enzyme, we have determined the structure of DCase from Agrobacterium sp. strain KNK712. RESULTS: The crystal structure of DCase has been determined to 1.7 A resolution. The enzyme forms a homotetramer and each monomer consists of a variant of the alpha + beta fold. The topology of the enzyme comprises a sandwich of parallel beta sheets surrounded by two layers of alpha helices, this topology has not been observed in other amidohydrolases such as the N-terminal nucleophile (Ntn) hydrolases. CONCLUSIONS: The catalytic center could be identified and consists of Glu46, Lys126 and Cys171. Cys171 was found to be the catalytic nucleophile, and its nucleophilic character appeared to be increased through general-base activation by Glu46. DCase shows only weak sequence similarity with a family of amidohydrolases, including beta-alanine synthase, aliphatic amidases and nitrilases, but might share highly conserved residues in a novel framework, which could provide a possible explanation for the catalytic mechanism for this family of enzymes.

Effects of maitake (Grifola frondosa) D-Fraction on the carcinoma angiogenesis.

We have reported that D-Fraction extracted from maitake (Grifola frondosa), activates immune competent cells, and indicates anti-tumor activities. The D-Fraction was observed to induce angiogenesis in vivo and to enhance the proliferation capability and migration capability of human vascular endothelial cell in vitro. The D-Fraction also increased plasma vascular endothelial growth factor (VEGF) concentration significantly. Also VEGF and TNF-alpha production by the activated peritoneal macrophages were enhanced. These results suggest that the anti-tumor activity of the D-Fraction is not only associated with the activation of the immuno-competent cells but also possibly related to the carcinoma angiogenesis induction.

Analysis of liquid accumulated in the distended capsular bag in early postoperative capsular block syndrome.

PURPOSE: To analyze the transparent liquid between the posterior lens capsule and the posterior chamber intraocular lens (PC IOL) in early postoperative capsular block syndrome and discuss the mechanism of posterior capsule distention. SETTING: Department of Ophthalmology, Tokyo University School of Medicine, and Tokyo Research Institute, Seikagaku Corporation, Tokyo, Japan. METHODS: This study evaluated 3 cases of capsular block syndrome presenting with transparent liquid in the distended capsular bag 1 day after cataract surgery. The transparent liquid material between the posterior capsule and PC IOL was aspirated and analyzed using high-performance liquid chromatography (HPLC). Also, sodium hyaluronate was diluted using a dialyzer to determine whether the aqueous humor was drawn into the capsular bag by an osmotic gradient across the capsule. RESULTS: The elution time of the samples was almost the same as that of sodium hyaluronate 1.0% (Healon) The concentration of the samples ranged from 3.29 to 9. 01 mg/mL by HPLC analysis. The sodium hyaluronate absorbed the physiological salt solutions through the dialyzer and expanded to 1. 9 times its original volume. CONCLUSIONS: These results indicate that the main ingredient of the transparent liquid in capsular bags is sodium hyaluronate and that the distention is caused by aqueous humor being drawn into the capsular bag by an osmotic gradient across the capsule when the capsulorhexis diameter is smaller than that of the PC IOL and by viscoelastic material retained and trapped in the bag intraoperatively.

Production of thermotolerant N-carbamyl-D-amino acid amidohydrolase by recombinant Escherichia coli.

A plasmid, pNT4553, was constructed for high level production of N-carbamyl-d-amino acid amidohydrolase (DCase), the thermostability of which has been improved by amino acid substitution. The DCase activity and the stability of the plasmid in the host cells were dependent on the Escherichia coli strains used. E. coli HB101 was the most suitable host strain among the 13 types of E. coli tested. E. coli HB101 exhibited the highest activity, i.e. 6.36 units/ml of culture broth in 2YT medium (1.6% tryptone, 1.0% yeast extract, and 0.5% NaCl, pH 7.0), and the plasmid was stably maintained by cultivation in 5 types of E. coli including HB101. Casamino acids, NZ-amine, peptone, and protein extract (a mixture of hydrolyzates of corn gluten, wheat gluten and soybean), were found to be suitable as natural nitrogen sources for both enzyme activity and growth. When cultivation was carried out in the presence of high concentrations of glycerol (6.5%) as the carbon source, and protein extract (3.0%) as the nitrogen source, in a small volume of the medium (20 ml of medium in a 500-ml shaking flask), in which the aeration level was estimated to be high, growth and activity reached OD550=63.8 (17.1 mg of dry cell weight/ml of culture broth) and 22.9 units/ml of culture broth, respectively. The economical hyperproduction of DCase using only inexpensive constituents for the medium was achieved.

The effect of maitake mushrooms on liver and serum lipids.

OBJECTIVE: To determine the efficacy of maitake mushrooms in inhibiting the elevation of liver and serum lipids in rats. DESIGN: Sprague-Dawley rats with hyperlipidemia were used to measure and compare the values of cholesterol, phospholipids, and triglycerides between cholesterol-fed rats and rats whose diets were fortified with 20% maitake mushroom dried powder. RESULTS: The values in maitake-fed rats were consistently less than those in the basic cholesterol-fed rats. The value of high-density lipoprotein cholesterol, which usually is decreased by taking high-cholesterol feed, maintained the level that it had at the beginning of the experiment. Weights of extirpated liver and epididymal fat pads were significantly less than those in the basic feed group. CONCLUSION: Our data suggest that maitake mushrooms have the ability to alter lipid metabolism by inhibiting both the accumulation of liver lipids and the elevation of serum lipids. Further studies are needed to elucidate the mechanism of activity of maitake mushrooms and to establish whether their action in humans is similar to that in the animal model tested here.

[Benign paroxysmal torticollis in infancy: case report].

Since Snyder reported first twelve cases of benign paroxysmal torticollis in infancy (BPT) in 1969, about 30 cases have been reported mainly in the North America and Europe, but not in Japan. The authors experienced such a case of one-year-old boy. The attack occurred at the age of 2 months for the first time and continued to repeat every 2 weeks periodically. The head tiltings were always noticed when he woke up in the morning and lasted for about 5 to 6 days, but they disappeared during sleep. He tilted his head to the lateral side more often to the left than to the right. There were associated symptoms and signs including irritability, vomiting, gait disturbance, and abnormal truncal posture during the attacks. He did not demonstrate any neurological abnormalities between the attacks. The study of cerebrospinal fluid, caloric test, computed tomography, electroencephalography and brainstem auditory evoked responses were also normal. The frequency of the attacks gradually decreased after the age of 2 years and disappeared at the age of 3 years, and he has not complained of severe headache or vertigo up to 6 years of age. This case is reported to emphasize that BPT has to be included in the differential diagnosis of acquired torticollis. And it is required to accumulate such cases to elucidate its etiological factors.

[Hemodynamic responses to nitrous oxide during isoflurane anesthesia in humans].

Isoflurane often produces tachycardia during clinical anesthesia. We examined the effect of a stepwise increase of isoflurane concentration on hemodynamic parameters in the absence or presence of nitrous oxide (N2O). After induction with thiamylal (3 mg.kg-1), isoflurane in oxygen or in 66% N2O-oxygen was administered with mask ventilation. Inspired isoflurane concentration was increased in a stepwise fashion (1, 2, 3 and 4%) every 5 minutes and manual ventilation was performed to maintain ETCO2 within 35-40 mmHg. Blood pressure (BP), heart rate (HR) and cardiac output (CO) were measured before and at every minute after isoflurane administration until 20 minutes. Systolic BP decreased gradually with increasing isoflurane concentration, but was transiently elevated for 3%. HR increased in a dose-related manner. CO decreased significantly at 1-3%. N2O and this seemed to magnify the isoflurane-induced decrease in BP and increase in HR at 1% and 2%. CO increased from baseline at 4%. Isoflurane tended to increase HR in a dose-related manner and induced a hyperdynamic response during rapid increasing of isoflurane concentration. This response may have beed caused by the irritating effect of isoflurane on the airways. Addition of N2O attenuated this response because it increases the speed of induction and the depth of anesthesia.