Isolation of urinary trypsin inhibitor-like inhibitor from human lung cancer tissue.

In the present study, a trypsin inhibitor was first extracted from lung cancer tissue and purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A final yield of 20 to 60 micrograms of inhibitor with a specific activity of 2040 units/mg of protein was obtained from 1 g of original lung cancer tissue. This inhibitor inhibited trypsin strongly, plasma kallikrein weakly, and plasmin more weakly, and its molecular weight was approximately 43,000 to 45,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its antigenicity was confirmed to be quite the same as that of human urinary trypsin inhibitor by double immunodiffusion, immunoelectrophoresis, and neutralization with anti-urinary trypsin inhibitor rabbit immunoglobulin.

GTP-dependent and -independent activation of superoxide producing NADPH oxidase in a neutrophil cell-free system.

GTP and GTP-gamma-S enhanced several-fold the NADPH-dependent superoxide production induced by sodium dodecyl sulfate in a cell-free system of pig neutrophils consisting of the membrane fraction and two cytosolic fractions separated by gel filtration. The enhanced activity was decreased by the addition of GDP in a dose-dependent manner, but 70% of the activity in the absence of GTP remained even at 1 mM GDP. Only one cytosol fraction besides the membrane fraction was required for the activation in the presence of GTP. The cytosol fraction was analyzed by chromatography on 2',5'-ADP agarose and two components responsible for the GTP-dependent and independent activation were separated. These results suggest that at least two pathways are available for the activation of superoxide production in the cell-free system of pig neutrophils.

Studies on the effect of plasminogen activator on the interaction between alpha 2-macroglobulin and plasmin.

Six different plasmins were prepared by incubating human plasminogen with various amounts of streptokinase or urokinase. It was confirmed that the six different plasmins possessed similar caseinolytic activities, and the inhibitory effects of a alpha 1-antitrypsin on caseinolytic activities of the six different plasmins were all the same. On the other hand, interactions between the six different plasmins and alpha 2-macroglobulin were complicated. Plasmins activated by cleavage of plasminogen were almost immediately or effectively inhibited by alpha 2-macroglobulin. However, plasmin activated by complex formation of plasminogen with streptokinase was not so immediately or effectively inhibited by alpha 2-macroglobulin. It was supposed that the difference between these two results on the interaction between plasmin and alpha 2-macroglobulin might be due to the difference in molecular form of plasmin. In the present study, it was also confirmed that streptokinase or urokinase, in free form in the reaction mixture, interfered with the interaction between plasmin and alpha 2-macroglobulin. The cause for such interference was discussed.

Studies on the activation mechanism of fibrinolytic enzyme system in plasma by human pancreatic elastase.

In the present study, the activation mechanism of fibrinolytic enzyme system in plasma by human pancreatic elastase was investigated. It was confirmed that human pancreatic elastase not only converted the co-existing plasminogen to low molecular weight-plasminogen which could be easily activated by the activator, but also inhibited alpha 2-macroglobulin and alpha 2-plasmin inhibitor which are antiactivators or fast reacting antiplasmins, and consequently, induced the activation of the fibrinolytic enzyme system in plasma.

Low molecular weight trypsin-plasmin inhibitors isolated from papain treated urinary trypsin inhibitor.

Papain treatment of human urinary trypsin inhibitor (UTI67; mol. wt. 43,000 by SDS-polyacrylamide gel electrophoresis, specific activity 1,897 U/mg protein) produced four new protease inhibitors, which were highly purified by gel chromatography on Sephadex G-100 and isoelectric focusing. The purified inhibitors (UTI26, UTI9-I, UTI9-II, and UTI9-III) were shown to be homogeneous by polyacrylamide disc gel electrophoresis, and had apparent molecular weights of 26,000, 9,000, 9,000, and 9,800, respectively, by sodium dodecyl sulfate gel electrophoresis. During enzymatic degradation of UTI67, the amino acid compositions changed to more basic, and the isoelectric point increased from pH 2.0 (UTI67) to pHs 4.4, 5.2, 6.6, and 8.3 (UTI26, UTI9-I, UTI9-II, UTI9-III), respectively. Both the parent and degraded inhibitors had anti-plasmin activity as well as antitrypsin and anti-chrymotrypsin activities. Much higher anti-plasmin/anti-trypsin and anti-plasmin/anti-chymotryspin activities were observed in the degraded inhibitors than in the parent UTI67. They competitively inhibited human plasmin with Ki values of 1.13 X 10(-7) - 2.12 X 10(-6) M (H-D-Val-Leu-Lys-pNA substrate). The reactions were very fast and the active site of the inhibitors of plasmin was thought to be different from that to trypsin or chymotrypsin.

Effect of 2',3'-dialdehyde NADPH on activation of superoxide-producing NADPH oxidase in a cell-free system of pig neutrophils.

We studied the effect of the 2',3'-dialdehyde derivative of NADPH on the activation of superoxide-producing oxidase in a cell-free system of pig neutrophils. The system consisted of a membrane fraction, two cytosolic fractions prepared by gel filtration, and arachidonic acid. Preincubation of one of the cytosolic fractions with the derivatives of NADPH and NADP+ caused the loss of its ability to activate the enzyme. The inactivation was effectively prevented by the addition of NADPH and NADP+. Neither the membrane fraction nor the other cytosolic fraction was affected by the derivatives. The results indicate that the NADPH binding component of the oxidase is present in the cytosolic fraction and may be translocated to the membrane fraction during the activation process in the cell-free system.

Studies on a kallikrein-kinin system in plasma of patients with acute pancreatitis: the preparation and characterization of a kallikrein-like enzyme in patient's plasma.

In a previous paper, the authors reported that the kallikrein-like activity was eluted in alpha 2-macroglobulin fractions when patient's plasma was fractionated with Sephadex G-200 gel filtration. In order to clarify the characteristics of the kallikrein-like enzyme, enzyme isolation methods were investigated. Success was attained by the addition of 1 % sodium-dodecyl-sulfate to alpha 2-macroglobulin fractions followed by G-200 chromatography. It was also confirmed that alpha 2-macroglobulin from which the enzyme was removed regained antiplasmin activity, and that some proteases other than the kallikrein-like enzyme were also bound to alpha 2-macroglobulin. The kallikrein-like enzyme isolated by sodium-dodecyl-sulfate-treatment was examined in respect of its molecular weight and its ability to be adsorbed on DEAE-cellulose and was found to possess a molecular weight approximating that of ovalbumin or human pancreatic kallikrein and a binding affinity for DEAE-cellulose. From these results, the authors speculate that during attacks of acute pancreatitis, the pancreas liberates kallikrein into the blood.

Studies on kallikrein-kinin system in plasma of patients with acute pancreatitis.

The fact that esterolytic activity is significantly elevated in plasma of patients with acute pancreatitis and that it correlates with the serum amylase level was confirmed. No kinin activity, however, was detected in plasma. To characterize the esterolytic activity, patient and normal plasmas were chromatographed on Sephadex G-200 and the esterolytic and the kinin-forming activities of each fraction were examined. In the present study, it was speculated that during attacks of acute pancreatitis, kallikrein with amylase might be liberated into blood from the pancreas. It was confirmed that almost all of the kallikrein liberated was combined with alpha1-macroglobulin, and alpha2-macroglobulin-bound kallikrein itself possessed kinin-forming activity.

Studies on the role of polymorphonuclear leukocyte granule elastase in Arthus reaction.

In the present study, attempts were made to clarify the role of an elastase-like enzyme in Arthus reaction by using rabbit-peritoneal polymorphonuclear leukocytes (PMNs). It was first confirmed that the injection of large amounts of PMNs with antibody can increase tissue damage in reversed Arthus reaction. The five various fractions, which were intact PMNs, whole homogenate, granule fraction, nuclei and cell debris, and membrane extract, were prepared from rabbit-peritoneal exudate. Each fraction was intracutaneously injected into the abdominal wall of rabbit. It was of particular interest that the tissue damage induced by membrane extract occurred earlier and was more severe than that caused by the others. Furthermore, membrane extract possessed remarkably higher caseinolytic and elastinolytic activities than the others. The membrane extract was partially purified by Sephadex G-100 column chromatography. The protein concentration of the elastase-like enzyme solution was 3.64 mg/ml, and its specific activity was 195 caseinolytic units and 0.45 elastinolytic units per milligram of protein. The cutaneous reaction by membrane extract was dependent on the concentration of this elastase-like enzyme and could be strongly inhibited by Elastatinal. From these results, the authors suggest that an elastase-like enzyme which originates in PMN granules is the key enzyme that promotes the severity of tissue damage in Arthus reaction.

Unusually late onset of cerebrospinal fluid rhinorrhea after head trauma.

Two cases of acute meningitis and cerebrospinal fluid rhinorrhea, in which the head trauma responsible occurred 10 and 30 years before, are presented. Intraoperatively, the brain parenchyma was found to be plugged into the fractured anterior fossa. By debridement and duraplasty from an intradural approach, both patients were cured. Several precipitating factors could be responsible for this unusually late reopening of the fistula. The possible accidental causes could be coughing or undetected microtraumas, but in the long run, atrophy of tissues and consequent changes in brain compliance with aging may play a role.

Effect of cepharanthine on neuro-humoral excitatory responses of gastric movement in dog.

In order to clarify the role of histamine in the neuro-humoral excitatory mechanism in the stomach, the effect of cepharanthine (an inhibitory agent of histamine release from mast cells) on excitatory response of gastric movement and increase in gastric mucosal histamine content caused by nerve stimulation (vagus and splanchnic nerves) or administration of tetragastrin were investigated in dogs. The animals were anesthetized with pentobarbital sodium and immobilized with gallamine triethiodide. The following results wee obtained. Increase of gastric mucosal histamine content caused by vagal and splanchnic nerve stimulation and administration of tetragastrin was inhibited by administration of cepharanthine. Cepharanthine inhibited the tonic contraction of the innervated antral pouch or isolated antral circular muscle strip caused by stimulation of vagus nerve. Tonic contraction of the innervated antral pouch caused by electrical stimulation of the splanchnic nerve after nicotinization of celiac and superior mesenteric ganglia was inhibited by administration of cepharanthine. Cepharanthine inhibited motor excitatory response of the denervated corpus pouch caused by stimulation of vagus and splanchnic nerves and by administration of tetragastrin. These results support the theory that cepharanthine inhibits the release of histamine from histamine-secreting cells in the gastric mucosa. It is suggested that histamine plays an important role in the neuro-humoral excitatory mechanism of gastric movement.

Pannus tissue at the cartilage-synovium junction in rheumatoid arthritis.

The cartilage-synovium junction of knees afflicted with rheumatoid arthritis was observed light microscopically using formalin-fixed, decalcified and immunohistochemically stained tissues. Decalcification had little or no influence on immunoreactivity for lysozyme and S-100 protein. All the specimens had pannus formation, which was classified into four types: A) cellular pannus with homogeneous cell pattern, B) cellular pannus of inflammatory cells, C) fibrous pannus with many fibrous bundles, D) fibrous pannus including round cells with scattered fibrous bundles. Type A pannus may be responsible for extensive cartilage degradation, and may occur at the first stage of pannus formation. Type B pannus may occur afterwards, and may be followed by type C pannus at a later stage. Type D pannus was found in two out of 19 specimens. Round cells in type D were positive for S-100 protein and lysozyme, and were probably chondrocytes. The findings indicated that chondrocytes were responsible for cartilage degradation and pannus formation.

Characteristics of stress fractures in young athletes under 20 years.

BACKGROUND: With the popularization of organized sports among adolescents and children, the clinical cases of stress fractures in young athletes have increased. This study was carried out to investigate the characteristics of stress fractures in young athletes. METHODS: During the 18-month period between January 1996 and June 1997, new clinical cases of stress fracture due to sports activities were collected from 39 hospitals in Japan. RESULTS: We analyzed 222 stress fractures in 208 athletes under 20 years of age (108 males: 14.4+/-2.2 years, 100 females: 15.2+/-1.5 years). Among these cases, the peak age of occurrence was 16. The most common sport causing stress fracture was basketball. The tibia was the most common location. About 70% of the patients participated in the respective sport for 6 or 7 days per week. High school students trained significantly more often than the primary and junior high school students (p<0.0001). There was a statistically significant difference in the return time to the sport between the cases who visited a hospital within 3 weeks and those who waited for more than 3 weeks after the onset of symptoms (return time, 10.4 vs 18.4 wks). Several cases visited a hospital beyond 8 weeks after the onset of symptoms regardless of the continuous pain of stress fracture, and their recovery was prolonged. CONCLUSIONS: It is important to educate young athletes that continuous pain lasting over 3 weeks is a warning signal to the body, and that early diagnosis leads to early recovery.

[Three cases of intracranial chordoma. Typical chordoma and chondroid chordoma].

Three cases of intracranial chordoma: 2 cases of typical chordoma and a case of chondroid chordoma, were reported and the clinico-pathological difference between these two types of chordoma was discussed. It is well known that typical chordoma is composed of physaliferous cells containing various quantities of intracytoplasmic mucin histologically, whereas chondroid chordoma as Heffelfinger reported in 1973 is composed of cellular area with scanty myxoid matrix and Chondroid matrix. Clinical course of the typical chordoma shows recurrence of the tumor within 2 years after surgical removal and radiation therapy in the majority of the cases, and the average survival range is said to be about 4.1 years, while chondroid chordoma takes benign course with average survival of 24.1 years after surgery and radiation therapy. One of our two patients with typical chordoma died from the recurrence two years after surgery and radiation therapy. However, the patients with chondroid chordoma showed no sign of recurrence over 2 years after subtotal removal and radiation. Surgical approach for the removal of the tumor was discussed as well. When the tumor is rather small and located at the upper clival region, transsphenoidal approach may be preferable, otherwise other approach should be chosen. When tumor has forward extension over the clivus, subtemporal approach will be most favorable with wider operative field. Identification of vessels and nerves around the tumor will be readily obtained by this approach.

[Effects of acupuncture therapy on low back pain and/or knee pain in elderly patients].

In April 1999, 75 elderly patients (mean age: 79 years old) with low back pain and/or knee pain visited an acupuncture and physical therapy unit in a geriatric hospital. A cross-sectional study was carried out in order to evaluate the effects of acupuncture therapy on low back pain and/or knee pain in elderly patients. Among them, 60 patients answered that their pain diminished following their therapy. The proportion of patients who were treated with acupuncture therapy were higher in these 60 patients than the other 15 patients (55.5% vs. 26.7%, p = 0.05). The result suggests that acupuncture therapy may be able to relieve low back pain and/or knee pain in elderly patients. However, 46% of the patients with acupuncture therapy were also treated with other types of physical therapy. Further studies should be recommended to confirm the effects of acupuncture therapy on low back pain and/or knee pain.

[Interaction of proteases and their inhibitors in the pathogenesis of pancreatitis].

It was confirmed that esterolytic activity was significantly elevated in plasma of patients with acute pancreatitis, which correlated better with the stage of the disease than serum amylase level. Using the several column chromatography procedures, pancreatic kallikrein, trypsin and pancreatic elastase were separated and purified from alpha 2-macroglobulin (alpha 2-M) fractions of patients plasma with acute pancreatitis. From this this result, it was confirmed that kallikrein was liberated into the blood stream from the pancreas during attacks of acute pancreatitis and the liberated kallikrein combined with alpha 2-M. Furthermore, the coexistence of trypsin is required for the complex formation of alpha 2-M and pancreatic kallikrein. It was speculated that alpha 2-M might be decomposed by the excessive amount of elastase, and consequently, might release all of its combining enzymes into the blood stream. In the present study, the activation mechanism of fibrinolytic enzyme system in plasma by human pancreatic elastase was investigated. Elastase not only converted the co-existing plasminogen to low molecular weight plasminogen which could be easily activated by the activators, but also inhibited alpha 2-M and alpha 2-plasmin inhibitor, and consequently, induced the activation of the fibrinolytic enzyme system in plasma. Furthermore, it was also confirmed that elastase could activate plasma kallikreinogen to kallikrein.

Successful closure of intractable tracheoesophageal fistula using a combination of a modified silicon stent and metallic stents.

Although surgery is the usual management strategy for acquired benign tracheoesophageal fistula, sometimes this approach is contraindicated or the patient declines surgical management. In this report, we describe a case of a patient with tracheoesophageal fistula at the level of the carina due to bronchial arterial infusion chemotherapy. Closure could not be achieved in response to multiple treatment strategies, including airway stenting, esophageal stenting, occlusion with microcoils, or cyanoacrylate glue. We subsequently achieved closure of this fistula through the combination of a modified silicon stent and metallic stents.