Increasing the Thermostable Sugar-1-Phosphate Nucleotidylyltransferase Activities of the Archaeal ST0452 Protein through Site Saturation Mutagenesis of the 97th Amino Acid Position

The ST0452 protein is a bifunctional protein exhibiting sugar-1-phosphate nucleotidylyltransferase (sugar-1-P NTase) and amino-sugar-1-phosphate acetyltransferase activities and was isolated from the thermophilic archaeon Sulfolobus tokodaii. Based on the previous observation that five single mutations increased ST0452 sugar-1-P NTase activity, nine double-mutant ST0452 proteins were generated with the intent of obtaining enzymes exhibiting a further increase in catalysis, but all showed less than 15% of the wild-type N-acetyl-d-glucosamine-1-phosphate uridyltransferase (GlcNAc-1-P UTase) activity. The Y97A mutant exhibited the highest activity of the single-mutant proteins, and thus site saturation mutagenesis of the 97th position (Tyr) was conducted. Six mutants showed both increased GlcNAc-1-P UTase and glucose-1-phosphate uridyltransferase activities, eight mutants showed only enhanced GlcNAc-1-P UTase activity, and six exhibited higher GlcNAc-1-P UTase activity than that of the Y97A mutant. Kinetic analyses of three typical mutants indicated that the increase in sugar-1-P NTase activity was mainly due to an increase in the apparent k(cat) value. We hypothesized that changing the 97th position (Tyr) to a smaller amino acid with similar electronic properties would increase activity, and thus the Tyr at the corresponding 103rd position of the Escherichia coli GlmU (EcGlmU) enzyme was replaced with the same residues. The Y103N mutant EcGlmU showed increased GlcNAc-1-P UTase activity, revealing that the Tyr at the 97th position of the ST0452 protein (103rd position in EcGlmU) plays an important role in catalysis. The present results provide useful information regarding how to improve the activity of natural enzymes and how to generate powerful enzymes for the industrial production of sugar nucleotides. IMPORTANCE It is typically difficult to increase enzymatic activity by introducing substitutions into a natural enzyme. However, it was previously found that the ST0452 protein, a thermostable enzyme from the thermophilic archaeon Sulfolobus tokodaii, exhibited increased activity following single amino acid substitutions of Ala. In this study, ST0452 proteins exhibiting a further increase in activity were created using a site saturation mutagenesis strategy at the 97th position. Kinetic analyses showed that the increased activities of the mutant proteins were principally due to increased apparent k(cat) values. These mutant proteins might suggest clues regarding the mechanism underlying the reaction process and provide very important information for the design of synthetic improved enzymes, and they can be used as powerful biocatalysts for the production of sugar nucleotide molecules. Moreover, this work generated useful proteins for three-dimensional structural analysis clarifying the processes underlying the regulation and mechanism of enzymatic activity

Progressing subglottic and tracheobronchial stenosis in a patient with CHARGE syndrome diagnosed in adulthood

AbstractA 33-year-old woman was admitted for a pseudocroup-like cough and wheezing after general anesthesia. Several months ago, she had undergone cardiac re-operation and turbinectomy, both of which had involved difficult intubations. Bronchoscopy indicated a pin-hall-like subglottic stenosis; therefore, emergency tracheotomy was performed. Six years later, a computed tomography scan demonstrated progressive stenosis of the entire circumference of the trachea and main bronchi. She died at 40 years. Her autopsy revealed marked tracheobronchial stenosis. She had many medical histories that had gone undiagnosed and had been clinically ill with only heart defects. She did not have coloboma but had microphthalmos, atresia choanae, retarded growth development, and deafness; thus, we diagnosed CHARGE syndrome that refers to multiple congenital anomalies, including airway abnormalities, which can lead to secondary complications such as traumatic stenosis after intubation. Physicians should have knowledge of this rare disease and should pay special attention to potential airway problems

Plasma level of D-dimer accompanying different types of gynecologic surgery and effects of prophylactic subcutaneous injection of heparin calcium

Background: The standard range of D-dimer level associated with each type of gynecologic surgery is required to note the occurrence of bleeding or thromboembolism.Methods: Plasma levels of D-dimer of patients who underwent different types of gynecologic surgery were measured on the Day of Preoperative Examination (DPE) and the first postoperative day (POD-1). Patients were classified by surgery type: hysterectomy for benign diseases or cervical intraepithelial neoplasia; hysterectomy for uterine cancer; surgery for ovarian cancer; laparoscopic surgery for a benign adnexal mass; laparotomy for a benign adnexal mass; laparotomic myomectomy; cervical conization; transcervical resection of an intrauterine mass; vaginal surgery for prolapse of a pelvic organ.Results: In each type of gynecologic surgery, plasma levels of D-dimer on POD-1 were higher than those on the DPE. Prophylactic subcutaneous injection of heparin calcium for patients who underwent surgery for endometrial cancer showed no significant difference in the plasma level of D-dimer on the sixth postoperative day (POD-6) and the plasma level of D-dimer on POD-6 was in the same level as those on POD-1.Conclusions: Plasma levels of D-dimer on POD-1 were higher than those on the DPE in each type of gynecologic surgery. The D-dimer level remained high even on POD-6, and not changed by prophylactic subcutaneous injection of heparin calcium.

High-speed processing for obtaining three-dimensional distance image and its application

A high-speed method of 3-D distance acquisition based on the triangulation principle is presented. This method uses conventional devices such as a CCD camera, a laser emitting semiconductor, and scanning mirrors; however, new circuits have been developed for detecting the position of spot image on the CCD. This development enables the high speed measurement and reduces the cost of the apparatus. Experiments showed that the apparatus and the method gave the practical measuring accuracy and speed, and it was found that the system is useful for image recognition. This method can easily display the stereoscopic image and cross-sectional figure of the object body. The method of real time processing has also been developed with the view to apply the device to the range finders for robots and blind persons</p