Acute oxidative stress modulates secretion and repetitive Ca2+ spiking in rat exocrine pancreas.

The effects of the oxidant tert-butylhydroperoxide (t-buOOH) on carbachol-stimulated pancreatic secretion in the vascularly perfused rat pancreas have been studied in parallel with [Ca2+]i signalling and amylase output in perifused rat pancreatic acinar cells. Perfusion of the pancreas with t-buOOH (0.1-1 mM) caused a rapid and irreversible inhibition of carbachol-stimulated (3x10-7 M) amylase and fluid secretion. Pre-perfusion of the pancreas with vitamin C and dithiothreitol or a cocktail of GSH and GSH-precursor amino acids provided only marginal protection against the deleterious effects of t-buOOH, even though GSH levels were elevated significantly. In perifused pancreatic acini, repetitive [Ca2+]i spikes evoked by carbachol (3x10-7 M) were sustained for 40 min. t-buOOH (1 mM) acutely increased the amplitude and duration of Ca2+ spikes, then attenuated Ca2+ spiking and subsequently caused a marked and sustained rise in [Ca2+]i. t-buOOH-induced alterations in carbachol-stimulated [Ca2+]i signalling and amylase release in perifused pancreatic acini were prevented by vitamin C. Although vitamin C restored impaired Ca2+ signalling and maintained amylase output in pancreatic acini, it seems likely that oxidative stress inhibits fluid secretion irreversibly in the intact pancreas, resulting in a loss of amylase output. Thus, perturbations in [Ca2+]i signalling may not fully explain the secretory block caused by oxidative stress in acute pancreatitis.

Identification of alpha- and beta-cells in intact isolated islets of Langerhans by their characteristic cytoplasmic Ca2+ concentration dynamics and immunocytochemical staining.

Ratiometric images of cytoplasmic Ca2+ concentration ([Ca2+]c) in individual cells were recorded simultaneously with a confocal ultraviolet-laser microscope in the Indo-1-loaded islets isolated from mice. After changes in [Ca2+]c in response to glucose or amino acids were recorded, the islet was fixed, permeabilized, and stained by the indirect immunofluorescence method against insulin or glucagon in situ; the individual cells were then identified in the focal plain identical to that used for the [Ca2+]c imaging. Almost all cells identified as insulin-positive (beta-cells) by their distinct immunofluorescence responded to the increase in glucose concentration from 3 to 11 mmol/l with an increase in [Ca2+]c. Major populations of cells (approximately 65%) identified as glucagon-positive (alpha-cells) responded to the addition of arginine (5-10 mmol/l) to 3 mmol/l glucose solution with an increase in [Ca2+]c. About half of the alpha-cells (47.6%) responded to the addition of alanine (5-10 mmol/l) to 3 mmol/l glucose solution with an increase in [Ca2+]c. In contrast, <13% of beta-cells responded to the addition of alanine (5-10 mmol/l) or arginine (5-10 mmol/l) to 3 mol/l glucose with an increase in [Ca2+]c. More than one-fourth of alpha-cells responded with an increase in [Ca2+]c when glucose concentration in perifusion solution was reduced from 11 to 0 mmol/l. These results indicate that [Ca2+]c changes in islet cells stimulated by glucose or amino acid were characteristic of the cell species, at least in the alpha- and beta-cell. This technique provides a useful tool to investigate not only the intracellular signal transduction but also the intercellular signal transmission in the intact islet.

Assessment of P-glycoprotein in patients with malignant bone and soft-tissue tumors using technetium-99m-MIBI scintigraphy.

UNLABELLED: Overexpression of P-glycoprotein (Pgp) has been detected in many malignant tumors including bone and soft-tissue tumors. Technetium-99m-MIBI has proved to be a transport substrate for Pgp. The purpose of our study was to explore 99mTc-MIBI as a functional imaging agent reflecting Pgp expression in malignant bone and soft-tissue tumors. METHODS: Technetium-99m-MIBI scintigraphy was performed in 30 patients with malignant bone and soft-tissue tumors. Radionuclide angiography with 99mTc-MIBI was done and, at 15 min and 3 hr postinjection of the radiopharmaceutical, imaging was performed. The 99mTc-MIBI uptake ratio was calculated by dividing the lesion count by the background count. The washout rate (WR) for 99mTc-MIBI was calculated by the following formula: WR = 100 x [(Te-Be)-(Td-Bd)]/(Te-Be) (%), where Te and Td = decay-corrected count density of the tumor in the 15-min and 3-hr images, respectively. Be and Bd = decay-corrected count density of the background in the 15-min and 3-hr images, respectively. The lesions were resected by open biopsy to obtain a histopathological diagnosis, and immunohistochemical staining was performed to detect Pgp. RESULTS: Twenty-four of 30 patients showed significant uptake at the 15-min image. In these 24 patients, the lesions with a high Pgp expression showed a similar 99mTc-MIBI perfusion index (3.00 +/- 1.04) and uptake ratio (2.05 +/- 0.58) at the 15-min image to those of lesions without a high Pgp expression (2.65 +/- 0.85 and 2.28 +/- 0.64, respectively). On delayed images, the 99mTc-MIBI uptake ratio was lower in patients with a high Pgp expression than in patients without a high Pgp expression (1.37 +/- 0.41 versus 1.87 +/- 0.39, p < 0.01). The washout ratio of 99mTc-MIBI was higher in patients with a high Pgp expression than in patients without a high Pgp expression (66% +/- 25% versus 29% +/- 18%, p < 0.001). None of the 6 patients without 99mTc-MIBI uptake at the 15-min imaging showed 201TI uptake, and only 2 had a high Pgp expression. CONCLUSION: In malignant bone and soft-tissue tumors, perfusion and initial 99mTc-MIBI uptake were not related to the Pgp expression; however, washout of 99mTc-MIBI from the tumor was related to Pgp expression. Technetium-99m-MIBI scintigraphy with washout analysis may be a useful method for the evaluation of Pgp overexpression and its function.

Formation and the inhibition of reaction plug in mated Drosophila--study of a primitive defense reaction.

Formation and inhibition of a reaction plug in the course of insemination reaction in Drosophila (Diptera) were investigated. When the reaction plug was formed in the vagina, fecundity was significantly reduced in interspecific crosses although the reaction plug did not affect the oviposition in intraspecific crosses. Some phenoloxidase blockers were found to be the effective inhibitors. The formation of the reaction plug was likely to be the consequence of polymerization and/or conformational change(s) of phenol-containing substance(s) involving nearly the same course of the melanization cascade reaction studied as a self-defense system in other insects.

Deleterious effect of null phenoloxidase mutation on the survival rate in Drosophila melanogaster.

The effect of null activity of phenoloxidase on the survival rate was investigated in mutants of Drosophila melanogaster. MoxGM95 and Dox-3KD95, structural genes for prophenoloxidase A1 and A3, were found in natural populations in the former Soviet Union, and affected the phenoloxidase activity in active A1 or A3, respectively. After linking the visible markers located on the second chromosome together with the variants, cross experiments were performed to make homozygote, rdo Dox-3KD95 pr C MoxGM95 wt. No double mutant had emerged. In the mutant, c MoxGM95 wt Pu2, the viability was greatly reduced. These results suggested that phenoloxidase and tyrosine-3-hydroxylase act as indispensable proteins to maintain life in Drosophila.

The full agonistic effect of recombinant 20 kDa human growth hormone (hGH) on CHO cells stably transfected with hGH receptor cDNA.

The agonistic effect of the recombinant 20 kilodalton human GH (20K-hGH) with authentic primary structure was studied using Chinese hamster ovary (CHO) cells stably transfected with hGH receptor (hGHR) cDNA and was compared with that of 22K-hGH. The binding affinities (dissociation constants) of 20K- and 22K-hGH were identical (0.41 +/- 0.11 nM and 0.41 +/- 0.04 nM, respectively). In addition, the two hGHs possessed the same potencies in activating the rat serine protease inhibitor (Spi) 2.1 gene promoter. 20K-hGH was similarly internalized as 22K-hGH but its internalization rate was a little slower than that of 22K-hGH. We also found that proliferation of CHO-hGHR cells stimulated by serum was remarkably inhibited by both hGHs to the same degree. In conclusion, both hGH isoforms exhibited the same binding affinities for hGHR and were potent enough to induce some hGHR-mediated cellular events. These suggest that 20K-hGH exerts a full agonistic activity for hGHR.

GH induced lipolysis stimulation in 3T3-L1 adipocytes stably expressing hGHR: analysis on signaling pathway and activity of 20K hGH.

We have constructed a cell line of 3T3-L1 which can efficiently express human GHR (3T3-L1-hGHR) after differentiation to adipocytes. The expressed hGHR was detected as two bands with approximate molecular sizes of 120K by Western analysis using hGHR specific monoclonal antibody. Maximum lipolytic activity induced by hGH in the 3T3-L1-hGHR was enhanced 10-fold as compared to that in 3T3-L1, suggesting that expressed hGHR is functionally active. Comparative analysis using bGH and hGH revealed that 70% of lipolysis stimulation by 1-10 ng/ml hGH could be attributed to hGHR-mediated response. Analyses on inhibition and phosphorylation of signaling molecules suggested that GH-induced lipolysis stimulation is dependent on gene expression and not mediated through PKA-, PKC-, PLA-, PLC-, nor MAPK-pathway but possibly through JAK-STATs pathway. Duration of STAT5 activation by hGH continued up to 48 h. We also revealed that 22 K hGH isoform, 20K hGH which has been reported as a weaker agonist for GH-induced lipolysis stimulation, possesses equipotent activity and shows stronger action in the presence of hGHBP as compared to 22 K hGH. Taken together we conclude that the hGH-induced lipolysis was not mediated through MAP-, PKA-, PKC-, nor PLA-pathway but might be mediated through STAT pathway and that 20K hGH might show higher lipolytic activity than 22 K hGH in adipose tissue that produces a large amount of GHBP.

Activation of prophenoloxidase with 2-propanol and other organic compounds in Drosophila melanogaster.

Activation with 2-propanol and other organic compounds of prophenoloxidase purified from pupae of Drosophila melanogaster was analyzed. A1, one of the two isozymes of the prophenoloxidase, could be activated with both an endogenous activating system and artificial organic compounds including alcohols. A1 was activated within 2 min after addition of 2-propanol. The phenoloxidase activity of A1, which had been activated with 2-propanol, decreased gradually by lowering the concentration of 2-propanol taking c 60 min to attain a low level, and the activity could be re-elevated at the re-introduction of 2-propanol. Thus the reversibility of the activation of A1 in response to the change of the concentration of 2-propanol in the activating mixture could be observed. Optimum concentration of 2-propanol for the rate of activation was 50%, optimum temperature was 30 degrees C and optimum pH was 7.5. The final level of the phenoloxidase activity, which had been activated with 2-propanol, was higher than that activated with the endogenous activating system. The activated state of A1 showed properties of a tyrosinase-type phenoloxidase. The results suggested that the activation of A1 with 2-propanol is caused by the reversible conformational change of the prophenoloxidase molecule.

Purification and characterization of prophenoloxidases from pupae of Drosophila melanogaster.

Two isoforms of prophenoloxidase were isolated from pupae of Oregon-R strain of Drosophila melanogaster. The purification procedure included ammonium sulfate fractionation, Sephacryl S-200 gel chromatography, DEAE-cellulose, and hydroxylapatite column chromatography. The two isoforms, A1 and A3, could be separated by ammonium sulfate fractionation. The isoelectric points of A1 and A3 were determined to be pH 5.8 and 6.7, respectively. The molecular weights of the monomers of A1 and A3 were estimated by SDS-PAGE to be 78 and 77 kDa, respectively. The native states of A1 and A3 are considered to be homodimeric, as judged by gel-filtration chromatography.

VIP- and PACAP-induced salivary chromogranin A secretion in the isolated perfused submandibular gland of rats.

In the study reported in this paper, sensitive ELISA for rat CgA was developed using synthetic rat CgA(359-389) as antigen, N alpha-biotinylated glycylglycyl rat CgA(359-389), and antirat CgA(359-389) serum for the measurement of CgA-LI in rat saliva. CgA-LI in rat submandibular tissues and saliva was characterized by both immunohistochemical and immunochemical methods. Using isolated perfused rat submandibular gland. VIP at 0.1-1.0 nM in the presence of 0.1 microM ACh was found to cause CgA-LI secretion, whereas neither PACAP-27 nor PACAP-38 showed any effect on CgA secretion.

Secretion of authentic 20-kDa human growth hormone (20K hGH) in Escherichia coli and properties of the purified product.

Using Bacillus amyloliquefaciens neutral protease gene (npr), we have constructed a secretion system of 20-kDA human growth hormone (20K hGH) in E. coli. The secretion-signal region from npr was modified inserting a fragment coding a 2Lys-5Leu cluster. In this system we found that co-expression of glutathione reductase remarkably increased accumulation level of 20K hGH in periplasm and confirmed that secreted 20K hGH was correctly processed. The recombinant 20K hGH was highly purified and subjected to analyses of physicochemical properties and biological activities which are still unclear and controversial due to difficulty in preparing the sample with authentic structure. The secreted recombinant product had authentic disulfide linkages and showed molecular weight of 20,270.5 +/- 3.7 (theoretical value, 20,269.9). The results suggest that the recombinant 20K hGH is a full agonist on rat growth promotion and lipolysis stimulation in isolated rat adipose tissues. In particular, the lipolysis-stimulating activity of 20K hGH was distinct as compared with that of 22K hGH under physiological concentration. Cell proliferation activity via prolactin-receptor in Nb-2 lymphoma was obviously low as compared with that of 22K hGH.

New modified activated partial thromboplastin time and prothrombin time methods using a synthetic chromogenic substrate in combination with diazotization.

A chromogenic substrate, H-D-Phe-Pip-Arg-pNA (S-2238) is a highly specific substrate to thrombin and releases p-nitroaniline (pNA) by the action of thrombin. We describe new modified APTT and PT methods using S-2238 in combination with the diazotization of pNA. In the modified APTT method, 100 microliter citrated plasma (diluted to 10-fold), 90 microliter 1 mM S-2238, 100 microliter 20 mM CaCl2 and 100 microliter Actin were mixed in an ice-bath, then incubated for 8 min at 37 degrees C. The reaction was stopped, and the generated pNA was diazotized by adding the following solutions sequentially: 975 microliter 0.04% sodium nitrite, 975 microliter 0.3% ammonium sulfamate and 975 microliter 0.07% N-(l-naphthyl)-ethylenediamine dihydrochloride. Diazotization changed pNA from yellow to pink. Then, absorbance at 545 nm was read, and values were expressed as thrombin units/ml plasma. In the modified PT method, 100 microliter citrated plasma (diluted to 20-fold), 90 microliter 1 mM S-2238 and 200 microliter tissue thromboplastin-C solution were mixed and processed as above. Correlations of the present modified APTT and APTT methods, and of modified PT and PT methods were significant (r = 0.426, p less than 0.01 and r = 0.561, p less than 0.01, respectively).

De novo deletions of p53 gene and wild-type p53 correlate with acquired cisplatin-resistance in human osteosarcoma OST cell line.

BACKGROUND: Initial p53 status is a useful determinant of chemoresistance or chemosensitivity of primary tumors, however, it remains unclear whether p53 status is a critical chemoresistant marker in tumors that acquire drug-resistance after the initiation of chemotherapy. We investigated the relationship between p53 status and the development of resistance to cisplatin in osteosarcoma cell lines. MATERIALS AND METHODS: Cisplatin-sensitive human osteosarcoma OST cells and acquired cisplatin-resistant OST/R cells derived from OST cells were used. Single-strand conformation polymorphism (SSCP) analysis of exons 5 to 8, and immunohistochemistry using anti-p53 antibodies were analyzed to detect mutations of p53. Fluorescence in situ hybridization (FISH) and enzyme immunoassay (EIA) were performed to detect deletions of p53. RESULTS: SSCP and immunohistochemistry revealed that both cell lines had wild-type p53 gene and protein. However, in OST/R cells, genomic instability of chromosome 17 and de novo deletion of the p53 gene located in chromosome 17p were detected by FISH. The constitutive levels of wild-type p53 protein measured by EIA were significantly lower in OST/R cells than in OST cells. Furthermore, p53 induction was lost in OST/R cells after cisplatin exposure. CONCLUSIONS: De novo deletions of the p53 gene and wild-type p53 were associated with the acquisition of cisplatin-resistance in osteosarcoma.

Establishment and characterization of an acquired cisplatin-resistant subline in a human osteosarcoma cell line.

A cisplatin (CDDP)-resistant human osteosarcoma cell line (OST/R) was established by continuous exposure to CDDP. OST/R cells proved to be 6.73 times more resistant to CDDP compared with parental OST cells, and showed cross-resistance to carboplatin (CBDCA). The mechanism of CDDP resistance was a significant decrease of intracellular platinum accumulation which was 40% of that in OST cells. OST/R cells were exposed to CDDP for 6 hours, the platinum was released from the cytoplasm of OST/R cells without reaching a state of equilibrium. DNA synthesis in OST/R cells was not inhibited by CDDP exposure, while in OST cells it was reduced by 50%. These data provide the first evidence that the increased efflux of platinum may play an important role in CDDP-resistance.

Caffeine-potentiated chemotherapy and conservative surgery for high-grade soft-tissue sarcoma.

We report here the results of preoperative and postoperative caffeine-potentiated chemotherapy and limb-sparing surgery for soft-tissue sarcomas. Thirty-six patients with histologically high-grade soft-tissue sarcomas were treated with caffeine-potentiated chemotherapy and conservative surgery (25 cases of limb-sparing surgery and 11 of local tumor excision). There were 13 patients with malignant fibrous histiocytoma (MFH), eight with synovial sarcoma, five with liposarcoma, four with malignant schwannoma, four with epithelioid sarcoma, one with leiomyosarcoma and one with extraskeletal chondrosarcoma. Nine patients were at stage III with lung metastasis and the other 27 at stage IIB without metastasis; 22 were male and 14 female with a mean age of 48 years, ranging from 16 to 77. For intra-arterial preoperative chemotherapy, we administered 2-5 courses of cisplatin (120 mg/m2), doxorubicin (30 mg/m2 x 2 days), and caffeine (1.5 g/m2 x 3 days) to 18 patients, and cisplatin and caffeine to the other 18. Although 15 patients had already undergone unplanned tumor excision at other hospitals before preoperative chemotherapy, all patients underwent definitive limb-sparing surgery after the preoperative chemotherapy. Surgical margins were wide for 28 patients, marginal for three and intralesional for five. Local tumor recurrence was seen in one patient with MFH and one with epithelioid sarcoma. Of the 27 stage IIB patients, lung metastasis newly developed in one with MFH, three with synovial sarcoma, two with malignant schwannoma and one with leiomyosarcoma. As for the effects of preoperative chemotherapy in the 33 eligible cases, radiographically confirmed complete response was seen in two patients, partial response in 20 and no response in 11. Histological response to this preoperative chemotherapy consisted of grade I (no response) in 14, grade II (50-90% necrosis) in four, grade III (> 90% necrosis) in eight, and grade IV (no viable cells) in seven cases. An overall objective response rate of 73% was obtained. With the mean follow-up period of 58 months (5-101 months), the overall 5-year cumulative survival rate ascertained with the Kaplan-Meier method was 63% and that of stage II patients 81%. Eight of the nine stage III patients died of metastatic disease within two and a half years from the beginning of the treatment. In conclusion, caffeine-potentiated chemotherapy and limb-sparing surgery brought good results for stage II nonmetastatic soft-tissue sarcomas. The problem of treatment for stage III metastatic soft-tissue sarcomas, however, remains unsolved.

Disappearance of Ewing's sarcoma following bacterial infection: a case report.

We report a patient with multiple metastases of Ewing's sarcoma in whom the tumor vanished after a bacterial infection. To the best of our knowledge, no comparable case with Ewing's sarcoma has been reported in the literature. The patient was a 17-year-old male who had an irregular destructive lesion of the left pelvis on radiologic examination. Pathologic examination of a biopsy specimen revealed Ewing's sarcoma. After the operation, a roentgenogram showed multiple spinal metastases with paraplegia. Despite initiation of chemotherapy, a subsequent bone scan showed several areas of increased uptake indicating multiple metastatic lesions. A fistula with purulent discharge opened at the operative site. While being treated with antibiotics and fistula irrigation, the fistula narrowed and his high fever subsided. During this period, radiologic examinations indicated that the multiple bone metastases had nearly disappeared. Nine years after the operation, the patient is alive without any evidence of tumor. We postulate that the antitumor activity in this patient resulted from the bacterial infection, and believe that this case supports continued consideration of immunotherapy for cancer.

Caffeine-potentiated radiochemotherapy and function-saving surgery for high-grade soft tissue sarcoma.

Caffeine, which has a DNA-repair inhibiting effect, enhances the cytocidal effects of anticancer drugs and radiation. We present a preliminary report on the results of a new treatment, "radiochemotherapy combined with caffeine" (K3 protocol), for high-grade soft tissue sarcomas. Seventeen patients with various high-grade soft tissue sarcomas were included in this study. Preoperatively, three to five courses of intra-arterial chemotherapy using cisplatin, caffeine and doxorubicin after radiation therapy were administered. Following the preoperative therapy, function-saving surgery was performed for all cases. Complete response was observed in six patients, partial response in six and no change in five. The effectiveness rate of caffeine-potentiated radiochemotherapy was therefore 71%. The histological response for radiochemotherapy was better than that for chemotherapy alone, that is, total tumor necrosis was identified in six patients and over 90% necrosis in another six. Complications resulting from the preoperative radiation comprised of serious inflammation in three patients and skin necrosis in another three. Twelve patients have remained free of disease, two patients are alive with disease and three have died of metastatic disease with a mean follow-up period of 36 months. There was no local tumor recurrence. These preliminary findings suggest that caffeine-potentiated radiochemotherapy contributed to a satisfactory local response and the success of function-saving surgery for high-grade soft tissue sarcomas.

Caffeine-assisted chemotherapy and minimized tumor excision for nonmetastatic osteosarcoma.

Osteosarcoma is usually treated with intensive preoperative and postoperative chemotherapy and wide tumor resection, resulting in a 60% to 70% 5-year survival rate. Caffeine has a DNA-repair inhibiting effect. We therefore investigated the impact of caffeine given in conjunction with chemotherapy and limb-sparing surgery on survival and local tumor control in patients with nonmetastatic, high-grade osteosarcoma. Twenty-two patients were given 3 to 5 preoperative courses of intra-arterial cisplatin (120 mg/m2, 1 to 2 hours) and caffeine (1.5 g/m2/day x 3 days) with or without doxorubicin (30 mg/m2/day x 2 days). Following this treatment, limb-sparing surgery was performed by means of intentional marginal excision aiming at preservation of important structures such as major neurovascular bundles, tendons, ligaments and the epiphysis. Three courses of cisplatin and doxorubicin combined with caffeine, and high-dose methotrexate with vincristine and citrovorum factor rescue were given intravenously as postoperative chemotherapy for 21 patients and three courses of high-dose methotrexate and combination of ifosfamide, etoposide and methotrexate for 1 patient. Following the preoperative chemotherapy, there were no viable tumor cells in 19 patients, only scattered foci of viable cells in 2 patients, and some areas of viable tumor cells in 1. The 21 patients with a good chemotherapeutic response on radiographs underwent minimized marginal excision. Functional evaluation of the affected limbs was excellent for 17 patients, good for 3, fair for 1, and poor for 1. No local tumor recurrence was seen in this series. Eighteen patients remain disease-free with a mean follow-up of 61 months. Two patients died of metastatic disease, 1 died of chemotherapy-related complications, and 1 died of unknown causes. The overall 5-year cumulative survival rate was 90%, and the 5-year event-free survival rate was 75%. Chemotherapeutic caffeine enhanced tumor necrosis and improved the success rate of limb-sparing surgery using marginal procedure without any adverse impact on survival. The results of our limited clinical trial appear to justify further prospective, multicenter randomized trials of the benefits of caffeine combined with chemotherapy for nonmetastatic osteosarcoma and other malignant neoplasms.

Limb salvage using distraction osteogenesis. A classification of the technique.

We report the results of distraction osteogenesis (callotasis) for the reconstruction of extensive defects after the excision of skeletal tumours in the limbs. Bone transport was performed in ten patients (five osteosarcomas and five giant-cell tumours), shortening-distraction in three (two osteosarcomas and one Ewing's sarcoma), and distraction osteogenesis combined with an intramedullary nail to reduce the time of external fixation in six (three osteosarcomas, two chondrosarcomas, and one malignant fibrous histiocytoma). The mean length of the defects after excision of the lesion was 8.4 cm. The mean external fixation index was 39.5 days/cm for the group treated by bone transport, 34.1 days/cm for the shortening-distraction group, and 24.0 days/cm for the group treated by distraction and an intramedullary nail. Functional evaluation gave excellent results in 12 patients, good in five and fair in two. There were ten complications in 19 patients, all of which were successfully treated. We also classified reconstruction using distraction osteogenesis into five types based on the location of the defects after resection of the tumour: type 1, diaphyseal; type 2, metaphyseal; type 3, epiphyseal; type 4, subarticular reconstruction; and type 5, arthrodesis. Our results suggest that reconstruction using distraction osteogenesis provides bone which will develop sufficient biomechanical strength and durability. It is beneficial in patients with an expectation of long-term survival and in growing children.

Production of the modified form of human plasminogen by alpha 2-macroglobulin-plasmin complexes.

It has been speculated that the modified form of plasminogen, a precursor of proteolytic enzyme plasmin in plasma, plays an important role in fibrinolysis in the blood. The present study was undertaken to examine the production by alpha 2-macroglobulin-plasmin complexes. alpha 2-Macroglobulin-plasmin complexes were purified from urokinase-activated plasma by affinity chromatography on lysine-Sepharose and gel filtration on Ultrogel AcA 22. The plasmin complex converted native plasminogen into the modified form more easily in the presence of epsilon-aminocaproic acid. The modification of native plasminogen by alpha 2-macroglobulin-bound plasmin was completely inhibited by aprotinin, and partly by soybean trypsin inhibitor. alpha 2-macroglobulin-bound plasmin produced modified plasminogen in human plasma where potent plasmin inhibitors exist, though the degree of production was small. The present results support the speculation of the important role of the modified form in vivo.