SAR analysis of the improved resonant cavity applicator with electrical shield and water bolus for deep tumors by a 3-D FEM.

This paper discusses the improvements of the re-entrant resonant cavity applicator, such as an electromagnetic shield and a water bolus for concentrating heating energy on deep tumors in an abdominal region of the human body. From our previous study, it was found that the proposed heating system using the resonant cavity applicator, was effective for heating brain tumors and also for heating other small objects. However, when heating the abdomen with the developed applicator, undesirable areas such as the neck, arm, hip and breast were heated. Therefore, we have improved the resonant cavity applicator to overcome these problems. First, a cylindrical shield made of an aluminum alloy was installed inside the cavity. It was designed to protect non-tumorous areas from concentrated electromagnetic fields. Second, in order to concentrate heating energy on deep tumors inside the human body, a water bolus was installed around the body. Third, the length of the lower inner electrode was changed to control the heating area. In this study, to evaluate the effectiveness of the proposed methods, specific absorption rate (SAR) distributions were calculated by FEM with the 3-D anatomical human body model reconstructed from MRI images. From these results, it was confirmed that the improved heating system was effective to non-invasively heat abdominal deep tumors.

Subcutaneous hematoma associated with manual cervical massage during carotid artery stenting. A case report.

We describe a patient with subcutaneous hematoma associated with manual cervical massage during carotid artery stenting.A 73-year-old man with left cervical carotid artery stenosis presented with left amaurosis fugax. We performed carotid artery stenting using distal embolic protection with balloon occlusion. Dual antiplatelet therapy was maintained in the periprocedural period and an anticoagulant agent was administered during the procedure. Because the aspiration catheter became entrapped by the stent, it did not reach the distal side of the stenotic lesion, and manual compression of the cervical region was therefore performed. Immediately afterwards, a subcutaneous hemorrhage occurred in the cervical region. There was no postoperative dyspnea due to enlargement of the hematoma, which was absorbed spontaneously.Cervical subcutaneous hematoma can occur in the cervical region due to cervical massage in patients who are receiving adjuvant antiplatelet therapy and anticoagulation therapy.

Heating properties of a new hyperthermia system for deep tumors without contact.

In this paper, heating properties of the proposed hyperthermia system for non-invasive treatment of deep tumors are discussed. Our heating system is composed of a large size resonant cavity applicator. In this heating method, a human body is placed between the two inner electrodes. It is heated by electromagnetic fields stimulated in the cavity without contact between the surface of the human body and the applicator. First, we presented the experimental results of heating a cylindrical agar phantom and a cylindrical fat-agar phantom using the proposed system. From the thermal images of the heated phantoms, the center of the agar was locally heated to maximum temperature. Second, we presented the experimental results of heating a mini pig. In the heating experiment, temperature measurements were performed by using fiber-optical thermometers inserted in four locations inside the mini pig. From the results, the deepest region of the liver was heated to the highest temperature 43.3 °C.

Protective effect of keishi-bukuryo-gan and its constituent medicinal plants against nitric oxide donor-induced neuronal death in cultured cerebellar granule cells.

Keishi-bukuryo-gan (Gui-Zhi-Fu-Ling-Wan) (KBG) is a traditional Chinese/Japanese medical (Kampo) formulation that has been administered to patients with "Oketsu" (blood stagnation) syndrome. In the process of neuronal cell death induced by brain ischemia, excessive generation of nitric oxide (NO) free radicals is implicated in the neurotoxicity. In the present study, we examined the protective effects of KBG and its constituent medicinal plants against NO donors, sodium nitroprusside (SNP) and 2,2'-(hydroxynitrosohydrazino)bis-ethanamine (NOC18)-induced neuronal death in cultured rat cerebellar granule cells (CGCs). MTT assay showed cell viability to be significantly increased by the addition of KBG extract (KBGE) (100 microg/ml), Cinnamomi Cortex extract (CCE) (3, 10 and 30 microg/ml), Paeoniae Radix extract (PRE) (100 microg/ml) and Moutan Cortex extract (MCE) (10 and 30 microg/ml) compared with exposure to SNP (30 microM, 24 h) only. Also, cell viability was significantly increased by the addition of KBGE (100 and 300 microg/ml), CCE (30 and 100 microg/ml), PRE (100 and 300 microg/ml) and MCE (30 and 100 microg/ml) compared with exposure to NOC 18 (100 microM, 48 h) only. Persicae Semen extract and Hoelen extract did not protect against NO donor-induced neuronal death. These results suggest that KBG has protective effect against NO-mediated neuronal death in cultured CGCs and that it is derived from Cinnamomi Cortex, Paeoniae Radix and Moutan Cortex.

Optimization of radioimmunotherapy interactions with hyperthermia.

The efficacy of radioimmunotherapy (RIT) employing radiolabelled monoclonal antibodies (MAb) is currently limited in most solid tumours. The combination of local hyperthermia (HT) with RIT has the potential to enhance tumour targeting of MAb; moreover, this approach may add an antitumour effect to radioresistant hypoxic and S-phase cells and may inhibit the cells from repairing sublethal damage or potentially lethal damage caused by ionizing radiation. There are distinct types of protocols in this combination. Hyperthermic temperature and timing relative to RIT administration appear to affect the efficacy of the combination therapy. Responses to heating at any particular condition are not always the same among different tumour types. There are many papers describing influence of HT on the biodistribution of radiolabelled MAb, but only limited information is currently available on 'therapeutic' outcomes regarding the dependency of combination protocols. A previous study suggested that the best therapeutic improvement would be achieved when HT was combined immediately after the administration of MAb, which significantly increases the radiation absorbed dose to tumours and produces a uniform intratumoural dose distribution. Further therapeutic investigation should be required to reach the optimal protocol of combining these two modalities.

Inhibitory effects of Choto-san (Diao-teng-san), and hooks and stems of Uncaria sinensis on free radical-induced lysis of rat red blood cells.

The present study is designed to test our hypothesis that the ingestion of Uncaria sinensis (US), the main medicinal plant of Choto-san (Diao-teng-san, CS), would protect red blood cell (RBC) membrane from free radical-induced oxidation if polyphenolics in US could be absorbed and circulated in blood. When incubated with RBC suspension, Choto-san extract (CSE) and Uncaria sinensis extract (USE) exhibited strong protection for RBC membrane against hemolysis induced by 2,2-azo-bis (2-amidinopropane) dihydrochloride (AAPH), an azo free-radical initiator. The inhibitory effect was dose-dependent at concentrations of 50 to 1000 microg/mL. Ingestion of 200 mg of USE was associated with a significant decrease in susceptibility of RBC to hemolysis in rats. Furthermore, caffeic acid, an antioxidative hydroxycinnamic acid, was identified in rat plasma after administration of URE.

Vitamin D receptor gene polymorphism detected by digestion with Apa I influences the parathyroid response to extracellular calcium in Japanese chronic dialysis patients.

BACKGROUND: To play its physiological role, 1,25(OH)2D3 must bind to a specific vitamin D receptor (VDR) in the nucleus. We have previously reported that VDR gene polymorphism influences the parathyroid function in patients with end-stage renal disease (ESRD). In the present study, we have investigated the relationship between the parathyroid responsiveness and VDR gene polymorphism, as detected by the Apa I restriction enzyme, by changing the concentration of Ca2+ in the dialysate. METHODS: 58 Japanese ESRD patients undergoing renal replacement therapy in our institution were evaluated. Genomic DNA was extracted from peripheral leukocytes and digested at the intron between exon 8 and exon 9 of the VDR gene using Apa I enzyme. Then alleles were classified into genotype A (undigested allele) and genotype a (digested allele). Extracellular ionized calcium ([Ca2+]e), serum phosphate, and intact parathyroid hormone (PTH) were measured before and after each hemodialysis (HD) session with dialysates having different concentrations of Ca2+ (1.5 or 1.25 mmol/l). The significance of differences in statistical analyses was defined within confidence limits of 5.0%. RESULTS: The AA, Aa, and aa genotypes were observed in 7/58 patients (12.1%), 23/58 patients (39.6%), and 28/58 patients (48.3%), respectively. The PTH reduction after HD with the 1.5-mmol/l Ca dialysate did not differ significantly between group AA+Aa and group aa. On the other hand, the PTH increase was significantly higher in group aa than in group AA+Aa after HD with the 1.25-mmol/l Ca dialysate (p = 0.0107), despite a similar PTH level before HD. Similarly, the percent increase of PTH after HD with the 1.25-mmol/l Ca dialysate was significantly higher (p = 0.0112) in group aa (50.2 +/- 9.4%) than in group AA+Aa (19.7 +/- 7.2%). There were no significant differences between the two groups in [Ca2+]e nor in serum phosphorus (Pi) before and after HD with either dialysate. Group AA+Aa and group aa did not show statistically significant differences in age, female/male ratio, ratio of diabetic nephropathy, or dialysis period. CONCLUSIONS: The study results showed that the patients in group aa were more sensitive to changes in [Ca2+]e than those in group AA+Aa. Moreover, they suggested that the VDR gene polymorphism may affect parathyroid responsiveness to changes in [Ca2+]e, which in turn may influence onset and progression of hyperparathyroidism in ESRD patients.

Effects of erbium, chromium:YSGG laser irradiation on root surface: morphological and atomic analytical studies.

OBJECTIVE: The purpose of this study was to investigate the morphological and atomic changes on the root surface by stereoscopy, field emission-scanning electron microscopy (FE-SEM), and energy dispersive X-ray spectroscopy (SEM-EDX) after erbium, chromium:yttrium, scandium, gallium, garnet (Er,Cr:YSGG) laser irradiation in vitro. SUMMARY BACKGROUND DATA: There have been few reports on morphological and atomic analytical study on root surface by Er,Cr:YSGG laser irradiation. METHODS: Eighteen extracted human premolar and molar teeth were irradiated on root surfaces at a vertical position with water-air spray by an Er,Cr:YSGG laser at the parameter of 5.0 W and 20 Hz for 5 sec while moving. The samples were then morphologically observed by stereoscopy and FE-SEM and examined atomic-analytically by SEM-EDX. RESULTS: Craters having rough but clean surfaces and no melting or carbonization were observed in the samples. An atomic analytical examination showed that the calcium ratio to phosphorus showed no significant changes between the control and irradiated areas (p > 0.01). CONCLUSIONS: These results showed that the Er,Cr:YSGG laser has a good cutting effect on root surface and causes no burning or melting after laser irradiation.

Preventive effect of tooth fracture by pulsed Nd:YAG laser irradiation with diamine silver fluoride solution.

OBJECTIVE: The purpose of the present study was to evaluate the preventive effect of pulsed Nd:YAG laser irradiation with 38% diamine silver fluoride [Ag(NH3)2F] solution for the fracture of endodontically treated teeth in vitro. BACKGROUND DATA: There have been no reports on the preventive effect of tooth fracture using Nd:YAG laser with Ag(NH3)2F solution. MATERIALS AND METHODS: Twenty-eight human extracted teeth were used in this study. The teeth were randomly classified into four groups: control group, where tooth surfaces were not submitted to any treatment; group 1, where tooth surfaces were coated with 38% Ag(NH3)2F solution; group 2, where tooth surfaces were coated with Ag(NH3)2F solution and irradiated by pulsed Nd:YAG laser for 2 sec; and group 3, where tooth surfaces were coated with Ag(NH3)2F solution and irradiated by pulsed Nd:YAG laser for 10 sec. After preparation, shear tests were performed and the maximum load for the fracture was measured. Results were analyzed using the Scheffe test, and difference at p < 0.05 was considered significant. RESULTS: The failure load for group 2 (mean, 182.5 kg) had the highest mean value and differed significantly from those for the control group (mean, 146.3 kg) and group 1 (mean, 147.1 kg; p < 0.05). The failure loads for groups 1 and 3 (mean, 150.0 kg) did not differ significantly from that for the control group (p > 0.05). CONCLUSION: The results show that the application of 38% Ag(NH3)2F solution followed by pulsed Nd:YAG laser irradiation for 2 sec is useful for prevention of tooth fracture at endodontically treated teeth.

Optimal timing of administration of hyperthermia in combined radioimmunotherapy.

Local hyperthermia (HT) may enhance the efficacy of radioimmunotherapy (RIT). However, the optimal timing of HT relative to administration of antibody is unknown. Human colon cancer xenografts (290 +/- 26 mm3) were treated with 4.63 MBq 131I-A7 monoclonal antibody (MAb) anti-Mr 45,000 glycoprotein antigen on colorectal cancer, and HT at 43 degrees C for 1 h was administered at: (A), 2 days after the 131I-A7 injection at the maximum 131I-A7 tumor accumulation (radiation); (B), soon after the 131I-A7 injection aiming to increase the tumor accumulation of 131I-A7 due to HT vascular effects; or (C), 2 days before the 131I-A7 injection in an attempt at injecting 131I-A7 when increased antigen expression could be expected. Specific growth delay (SGD) of tumors was calculated as (Tqtreat-Tqcontrol)/Tqcontrol where Tq was tumor quadrupling time. The biodistribution and intratumoral distribution of 131I-A7 were investigated to explore the mechanism of tumor response among the different HT regimens. HT alone produced some antitumor effect (SGD 1.90 +/- 0.26), which was less effective than RIT (3.11 +/- 0.50). HT soon after 131I-A7 RIT (B) significantly enhanced RIT efficacy (6.57 +/- 0.51, p < 0.0001) whereas neither HT at 2 days after RIT (A) nor at 2 days before RIT (C) did so. Biodistribution study revealed that HT soon after RIT (B) increased the tumor radiation absorbed dose by a factor of 2.4, while HT after RIT (A) did not increase radiation dose and HT before RIT (C) decreased it. Radioluminograms of tumor sections indicated that HT soon after RIT (B) improved the uniformity of 131I-A7 distribution whereas HT after RIT (A) did not and HT before RIT (C) diminished the uniformity of A7 distribution. In conclusion, the best therapeutic efficacy was obtained when HT was combined soon after the initiation of RIT with 131I-A7. The increased tumor radiation absorbed dose and the uniform intratumoral distribution of 131I-A7 were important factors underlying this improvement, and the additive cytotoxicity of HT is suspected to some extent. HT-induced radiosensitization of tumor was not apparent in this model when HT was given 2 days after 131I-A7 MAb.

Transcriptional regulation of the CLC-K1 promoter by myc-associated zinc finger protein and kidney-enriched Krüppel-like factor, a novel zinc finger repressor.

The expression of CLC-K1 and CLC-K2, two kidney-specific CLC chloride channels, is transcriptionally regulated on a tissue-specific basis. Previous studies have shown that a GA element near their transcriptional start sites is important for basal and cell-specific activities of the CLC-K1 and CLC-K2 gene promoters. To identify the GA-binding proteins, the human kidney cDNA library was screened by a yeast one-hybrid system. A novel member of the Cys2-His2 zinc finger gene designated KKLF (for "kidney-enriched Krüppel-like factor") and the previously isolated MAZ (for "myc-associated zinc finger protein") were cloned. KKLF was found to be abundantly expressed in the liver, kidneys, heart, and skeletal muscle, and immunohistochemistry revealed the nuclear localization of KKLF protein in interstitial cells in heart and skeletal muscle, stellate cells, and fibroblasts in the liver. In the kidneys, KKLF protein was localized in interstitial cells, mesangial cells, and nephron segments, where CLC-K1 and CLC-K2 were not expressed. A gel mobility shift assay revealed sequence-specific binding of recombinant KKLF and MAZ proteins to the CLC-K1 GA element, and the fine-mutation assay clarified that the consensus sequence for the KKLF binding site was GGGGNGGNG. In a transient-transfection experiment, MAZ had a strong activating effect on transcription of the CLC-K1-luciferase reporter gene. On the other hand, KKLF coexpression with MAZ appeared to block the activating effect of MAZ. These results suggest that a novel set of zinc finger proteins may help regulate the strict tissue- and nephron segment-specific expression of the CLC-K1 and CLC-K2 channel genes through their GA cis element.

TOK-1, a novel p21Cip1-binding protein that cooperatively enhances p21-dependent inhibitory activity toward CDK2 kinase.

A p21(Cip1/Waf1/Sdi1) is known to act as a negative cell-cycle regulator by inhibiting kinase activity of a variety of cyclin-dependent kinases. In addition to binding of the cyclin-dependent kinase to the N-terminal region of p21, p21 is also bound at its C-terminal region by proliferating cell nuclear antigen (PCNA), SET/TAF1, and calmodulin, indicating the versatile function of p21. In this study, we cloned cDNA encoding a novel protein named TOK-1 as a p21 C-terminal-binding protein by a two-hybrid system. Two splicing isoforms of TOK-1, TOK-1alpha and TOK-1beta, comprising 322 and 314 amino acids, respectively, were co-localized with p21 in nuclei and showed a similar expression profile to that of p21 in human tissues. TOK-1alpha, but not TOK-1beta, directly bound to the C-terminal proximal region of p21, and both were expressed at the G(1)/S boundary of the cell cycle. TOK-1alpha also preferentially bound to an active form of cyclin-dependent kinase 2 (CDK2) via p21, and these made a ternary complex in human cells. Furthermore, the results of three different types of experiments showed that TOK-1alpha enhanced the inhibitory activity of p21 toward histone H1 kinase activity of CDK2. TOK-1alpha is thus thought to be a new type of CDK2 modulator.

Enhanced efficacy of radioimmunotherapy combined with systemic chemotherapy and local hyperthermia in xenograft model.

We previously found that the efficacy of radioimmunotherapy (RIT) with (131)I-A7, an IgG(1) against M(r) 45000 glycoprotein on colon cancer, was enhanced by local hyperthermia (HT) or chemotherapy with 5-fluorouracil (5-FU). In this study, we aimed to further enhance its efficacy by combining these three modalities. Human colon cancer xenografts (146 x 12 mm(3)) in Balb / c nu / nu female mice were treated with 9.25 MBq (131)I-A7 i.v. combined with HT (43 degrees C for 1 h) and 5-FU (30 mg / kg / day i.p. for 5 days). Tumor growth delay, (Tq(treated) - Tq(control) )/ Tq(control) where Tq is tumor quadrupling time, in mice treated with RIT + HT + 5-FU was improved to 12.7 from 5.90, 7.55 and 10.1 with RIT alone, RIT + 5-FU and RIT + HT, respectively. Complete response was observed in 4 out of 8 tumors with RIT + HT + 5-FU and 3 out of 10 with RIT + HT. No tumor showed complete response with RIT + 5-FU or RIT alone. 5-FU slightly increased myelotoxicity of RIT, but HT did not affect it. Body weight loss was not enhanced by the combination. These results indicate that the combination of three modalities is a feasible approach to enhance the antitumor efficacy of RIT without serious increase of toxicity.

Permeability of the root canal wall and occlusion of dentinal tubules by Ag(NH3)2F: a comparison of combined use with pulsed Nd:YAG laser or iontophoresis.

OBJECTIVE: We used SEM-EDX and the Vickers hardness test to compare the penetration and hardness of silver resulting from use of either (i) pulsed Nd:YAG laser or (ii) iontophoresis, after root canal wall shaping using the standard method of coating with 38% Ag(NH3)2F solution. SUMMARY BACKGROUND DATA: There have not been any reports of penetration and hardness following the application of Ag(NH3)2F solution together with laser or iontophoresis. METHODS: We used 21 extracted human single-rooted teeth randomly divided into three groups. Group 1 was coated with Ag(NH3)2F, Group 2 was irradiated with a Nd:YAG laser after coating with Ag(NH3)2F solution, and Group 3 was iontophoresised after coating. Then we observed the permeability of silver through the root canal wall using SEM-EDX. RESULTS: The results show that iontophoresis after coating with Ag(NH3)2F solution (Group 3) resulted in the greatest and deepest penetration of silver into the root canal wall. There was no significant difference between teeth in Groups 1 and 2. For the hardness test, the 21 teeth were tested using SEM-EDX test, 7 untreated teeth were used as a control. The results show that Group 2 (laser treatment) teeth were the hardest. CONCLUSION: We therefore propose that root canals should be treated using irradiation with an Nd:YAG laser that has been coated with Ag(NH3)2F solution and that this method provides better results than either iontophoresis after coating, or coating alone.

Combination radioimmunotherapy with local hyperthermia: increased delivery of radioimmunoconjugate by vascular effect and its retention by increased antigen expression in colon cancer xenografts.

Hyperthermia (HT) may increase tumor targeting of a radiolabeled antibody by its effects on tumor vasculature and antigen expression. Expression of a 45-kDa glycoprotein antigen on LS180 colon cancer cells was 2.7-fold enhanced 2 days after heating at 43 degrees C for 1 h. Preferential tumor accumulation of 125I-A7 recognizing this antigen was doubled and the antitumor effect of 131I-A7 was significantly improved by HT. Hyperthermia also increased tumor uptake of an irrelevant antibody but its radioactivity was rapidly cleared. These results indicate that HT increased the initial delivery of an antibody to a tumor by its vascular effect, and radioactivity was retained in tumors by increased specific binding, resulting in a better radioimmunotherapy outcome.

Effects of an anti-angiogenic agent, TNP-470, on the growth of oral squamous cell carcinomas.

Anti-tumor effects of an anti-angiogenic agent, TNP-470, on the growth of oral squamous cell carcinomas (SCCs) were evaluated in vivo and in vitro. The growth of an oral SCC cell line, HSC-2, inoculated subcutaneously in severe combined immuno-deficiency (SCID) mice was inhibited in a dose-dependent manner by the treatment with this agent. A reduction of microvessels surrounding tumor tissues treated with TNP-470 was observed by immunohistochemistry. Significant side-effects were not observed except for weight loss during the period of treatment with high dose (50 mg/kg) of TNP-470. The direct effects of TNP-470 on oral SCC cell lines were also evaluated in culture. The growth of all eight SCC cell lines tested was inhibited by TNP-470, but the sensitivity of the oral SCC cell lines to TNP-470 was about 1700 times less than that of endothelial cells. These results suggest that TNP-470 inhibits the growth of oral SCC by anti-angiogenic activities and that it may be effective as a new therapy of oral cancer.

A comparative study on the effects of three types of laser irradiation at the apical stop and apical leakage after obturation.

OBJECTIVE: The purposes of this study were to determine the degree of apical leakage using a dye penetration method after argon, pulsed Nd:YAG with or without black ink, or Er:YAG laser treatment and obturation, and to compare the results of three types of laser irradiation in vitro. SUMMARY BACKGROUND DATA: There have been only a few reports of the laser effect on apical leakage after laser treatment and obturation. METHODS: Seventy-five human extracted single root teeth were divided into five groups of 15 teeth each and prepared up to a #60 K-file size. One group served as the control and was not irradiated. The other four groups were irradiated with an argon laser at 0.3 W for 3 seconds at continuous mode, pulsed Nd:YAG laser at 2 W and 20 Hz for 4 seconds with or without black ink, and Er:YAG laser at 50 mJ per pulse and 4 Hz for 5 seconds. In each group, ten teeth for the apical leakage study were obturated and immersed in rhodamine B solution for 48 hours at 37 degrees C, and the others were used for the observation by scanning electron microscopy (SEM). All teeth were longitudinally bisected and observed by stereoscopy or SEM. RESULTS: After pulsed Nd:YAG laser treatment with black ink and obturation, the apical leakage degree (mean score: 0) was reduced compared to that in the control (mean score: 1.2) significantly (p < 0.05). SEM findings showed that specimens irradiated by pulsed Nd:YAG laser with black ink presented clean root canal walls with debris and smear layer evaporated, melted, fused, and recrystallized in most cases. CONCLUSION: These results suggest that pulsed Nd:YAG laser irradiation with black ink at the apical stop is useful for the reduction of apical leakage after obturation.

[A randomized controlled study of total parenteral nutrition and enteral nutrition by elemental and polymeric diet as primary therapy in active phase of Crohn's disease].

We performed randomized controlled study to compare the short-term therapeutic effect of total parenteral nutrition (TPN), elemental diet (ED) and polymeric diet (PD) given as primary therapy in active phase of Crohn's disease. In hospital for Crohn's disease, twenty-eight patients were given nutritional therapy: 9 patients by TPN, 10 by ED, and 9 by PD. Nutritional state, inflammatory reactions, disease activity and clinical remission rate were assessed two weeks and four weeks after treatment, and morphological findings were assessed before and after each nutritional therapy by radiographic and colonoscopic findings. Inflammatory reactions were more effectively controlled by TPN and ED than by PD, and early improvement achieved by TPN and ED was especially note-worthy. Clinical remission rate after treatment by TPN was highest in three types of nutritional approach, but no significant difference was seen at any point. In nutritional state, disease activity and morphological findings, comparable changes were effected without preference. These results suggest that nutritional therapy by total parenteral nutrition and elemental diet is superior to polymeric diet for treating active phase of Crohn's disease with marked inflammatory reactions.

Overproduction of DnaJ in Escherichia coli improves in vivo solubility of the recombinant fish-derived transglutaminase.

The overexpression of red sea bream (Pagrus major) transglutaminase (TGase, E.C. 2.3.2.13) in Escherichia coli mostly leads to the accumulation of biologically inactive enzyme. Although the solubility of the gene products could be improved by cultivation at a lower temperature (26-28 degrees C), most of the synthesized TGase was still in the form of insoluble aggregates. The effects of overproduction of molecular chaperones on the intracellular solubility of newly produced recombinant TGase were examined. The overexpression of dnaK or groES/EL did not improve solubility. However, DnaJ greatly increased the solubility of the recombinant TGase, resulting in active enzyme in the presence of calcium ions. Co-expression of dnaK along with dnaJ further increased the content of soluble TGase. Under our experimental conditions, supplementation with both DnaJ and DnaK elevated the TGase activity in the producer cells by roughly 4-fold, compared with the control strain cultured at 30 degrees C. Thus, we found that DnaJ is important in controlling the solubility of protein overproduced in E. coli.