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The heterologous expression of the laccase gene from Trametes versicolor and Gaeumannomyces graminis was evaluated in the yeasts Pichia pastoris and Cryptococcus sp. S-2. The expression levels of both laccase genes in Cryptococcus sp. S-2 were considerably higher than those in P. pastoris. The codon usage of Cryptococcus sp. S-2 as well as the GC content were similar to those of T. versicolor and G. graminis. These results suggest that using a host with a similar codon usage for the expressed gene may improve protein expression. The use of Cryptococcus sp. S-2 as a host may be advantageous for the heterologous expression of genes with high GC content. Moreover, this yeast provides the same advantages as P. pastoris for the production of recombinant proteins, such as growth on minimal medium, capacity for high-density growth during fermentation, and capability for post-translational modifications. Therefore, we propose that Cryptococcus sp. S-2 be used as an expression host to improve enzyme production levels when other hosts have not yielded good results., (Copyright © 2012 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Yeast host-vector systems have been very successful in expressing recombinant proteins. However, because there are some proteins that cannot be expressed with existing systems, there is a need for new yeast expression systems. Here we describe a new host-vector system based on the basidiomycetous yeast Cryptococcus sp. strain S-2 (S-2). Two advantages of S-2 are that it naturally produces some very useful enzymes, so it would be a good system for expressing multiple copies of some of its genes, and that, it is a nonhazardous species. The orotate phosphoribosyltransferase (OPRTase, EC 2.4.2.10) gene (URA5) was selected as a selectable marker for transformation in the new host-vector system. URA5 was isolated and introduced into a uracil auxotroph of S-2 by electroporation. To demonstrate the S-2 system, we selected one of its unique enzymes, a plastic-degrading cutinase-like enzyme (CLE). We were able to insert multiple copies of the CLE gene (CLE1) into the chromosomes in a high fraction of the targeted cells. Under optimal conditions, one transformant exhibited 3.5 times higher CLE activity than the wild type. Expression vectors, including an inducible promoter (the promoter for the xylanase or α-amylase gene), were constructed for recombinant protein production, and green fluorescent protein was expressed under the control of these promoters. The xylanase promoter was more tightly controlled. Furthermore, putting CLE1 under the control of the xylanase promoter, which is induced by xylose, increased CLE activity of the culture medium to approximately 15 times greater than that of the wild type.

Periodontal regeneration has been challenged with chemical reagents and/or biological approaches, however, there is still no sufficient technique that can regenerate complete periodontium, including alveolar bone, cementum, and well-oriented collagen fibers. The purpose of this study was to examine multi-layered sheets of periodontal ligament (PDL)-derived cells for periodontal regeneration. Canine PDL cells were isolated enzymatically and expanded in vitro. The cell population contained cells capable of making single cell-derived colonies at an approximately 20% frequency. Expression of mRNA of periodontal marker genes, S100 calcium binding protein A4 and periostin, was observed. Alkaline phosphatase activity and gene expression of both osteoblastic/cementoblastic and periodontal markers were upregulated by osteoinductive medium. Then, three-layered PDL cell sheets supported with woven polyglycolic acid were transplanted to dental root surfaces having three-wall periodontal defects in an autologous manner, and bone defects were filled with porous beta-tricalcium phosphate. Cell sheet transplantation regenerated both new bone and cementum connecting with well-oriented collagen fibers, while only limited bone regeneration was observed in control group where cell sheet transplantation was eliminated. These results suggest that PDL cells have multiple differentiation properties to regenerate periodontal tissues comprising hard and soft tissues. PDL cell sheet transplantation should prove useful for periodontal regeneration in clinical settings.

Although the measurement of serum haptoglobin (S-Hp) is of great use for evaluation of intravascular hemolysis, it is not applicable in patients with mechanical prosthetic valves because S-Hp is virtually absent. We administered haptoglobin preparation to 10 patients with Björk-Shiley mitral prosthetic valves and 10 patients with the same aortic prosthetic valves. Serum haptoglobin levels were measured periodically afterwards. The maximum haptoglobin levels (Hp (max)), serum, haptoglobin reducing rate ((Hp-delta Hp)/delta t) and expected haptoglobin disappearing time (hours) were obtained from the subsequent samples. The screening studies which were performed at the same time were not predictors of difference in the 2 groups. On the other hand, serum haptoglobin reducing rate and expected haptoglobin disappearing time indicated that hemolysis is higher in patients with an aortic prosthetic valve than with a mitral prosthetic valve. This haptoglobin administration test seems to be useful for the comparative examination of the intravascular hemolysis caused by the difference in the position of the prosthetic valve.

A knitted Dacron velour externally supported prosthesis was broken in two pieces at the supported portion 3 years after femoroposterior tibial bypass grafting. This type of graft problem has never been reported. Details of the clinical course, angiogram, CT scan, and scanning electron micrograph of a portion of the graft are shown. The externally supported Dacron prosthesis broke into two pieces along the broken coil of the middle portion. A possible cause of this failure may have been due to damage of the Dacron fibers in the process of heat fusing the coil. This presentation emphasizes the possibility of this rare externally supported graft complication, and the necessity for careful follow-up not only of the graft patency but also for the mechanical defects of the implanted externally supported Dacron graft itself.

Two hundred and seventy-eight patients with atrial septal defect of secundum type were operated on surgical closure of the defect. The patients were divided into 5 groups according to age at surgery, and preoperative complaints and laboratory findings were analyzed to evaluate natural history. As year passed, incidence of complaints increased. Cardiothoracic ratio (CTR), systolic pressure of the right ventricle (PRVS), end-diastolic pressure of the right ventricle (RVEDP), mean pressure of the right atrium (PRAm), ratio of pulmonary to systemic systolic pressure (PPA/PS), right ventricular dimension (RVD) and left atrial dimension (LAD) increased significantly, while frontal axis of the QRS complex, RV1, SV1 +RV5 and LVD decreased. No chronological changes were seen in ratio of pulmonary to systemic blood flow (QPA/QS) and ratio of pulmonary to systemic vascular resistance (RPA/RS). The first and second decades of life were considered to be stable stages of the disease. Postoperatively, changes in the above-mentioned parameters were compared in each age group, and effects of surgical repair on natural history were evaluated. Of 265 survivors, 168 were followed-up for more than one year, the longest period being 22 years. CTR, frontal axis, RV1, PRVS, RVEDP, PPA/PS and RVD decreased significantly, while SV1 + RV5, RPA/RS and LVD increased. No changes were seen in LAD. Surgical effects appeared most significantly in the first decade and least in the fifth or more. From these findings it would be concluded that corrective surgery should be best carried out in the first decade, at latest in the second, though not to be contraindicated by age alone. Otherwise, postoperative improvement of parameters delays or hemodynamical abnormalities may persist regardless of considerable clinical improvement.