A cysteine-activated protease isolated from Todarodes pacificus squid degrades collagen below its denaturation temperature

Collagen purified from the mantle muscle of the Japanese common squid, Todarodes pacificus, showed autodegradation during incubation under acidic conditions at 25 degrees C, without the addition of exogenous enzymes. This suggests that the collagenolytic proteases bind to collagen tightly through the steps of collagen preparation. Collagenolytic activity also was detected in a crude extract of mantle muscle, and leupeptin and E-64 were observed to inhibit collagenolytic activity within the collagen fraction and muscle extract. We purified these collagenolytic cysteine proteases by leupeptin column chromatography and cellulose acetate membrane electrophoresis. Optimal enzymatic activity was observed at pH 3.5, and collagenolytic activity was completely suppressed at neutral or alkaline pH. The purified enzymes were 28 kDa and 25 kDa in size, and both had gelatinolytic activity, as detected by gelatin zymography, and cut the specific site of denatured collagen alpha chain. The purified enzymes degraded squid collagen at 25 degrees C, which is 2.5 degrees lower than the temperature at which squid collagen normally denatures; however, the proteases were ineffective at 20 degrees C. Interestingly, the isolated proteases were capable of digesting both squid and bovine gelatin. In this article, we describe collagenolytic cysteine proteases that bind to the collagen of Todarodes pacificus, thereby digesting it by attacking microunfolding regions generated by incubation 2-3 degrees C below the denaturation temperature.     

Phospholipid transmethylation and choline phosphotransferase in microsomal fraction of human diseased liver

The activities of three enzymes catalyzing the production or degradation of phosphatidylcholine, a major structural phospholipid of cell membranes, were assessed in hepatocyte membrane microsomal preparation from patients with various types of liver disease. Choline phosphotransferase activity of preparation from patients with chronic aggressive, chronic active, chronic persistent, alcoholic hepatitis and cirrhosis accompanied by marked necrosis and relatively slight fibrosis was markedly decreased, compared with normal liver; the activity from patients with fatty liver and chronic inactive hepatitis was slightly decreased. Specimens from patients with acute transient hepatitis were not significantly different from normal. Methyltransferase and phospholipase A2 activities tended to parallel that of choline phosphotransferase, although the degree of changes was generally less marked. Our studies indicate that enzyme activities that are critical for hepatic cell membrane integrity and activity are attenuated in liver specimens from patients with disease in which there is marked hepatic cell necrosis.     

Fetal diaphragmatic hernia: prenatal evaluation of lung hypoplasia and effects of immediate operation

The outcome of fetuses with diaphragmatic hernia (CDH) has been reported to be related to the severity of lung hypoplasia. As an index of pulmonary hypoplasia, we attempted to measure the lung-thorax transverse area ratio (L/T) using ultrasonic echography in eight fetuses with left-sided CDH. Two cases with L/T more than 0.28 (controls: 0.52±0.04) were transported postnatally and recovered after early operation without episodes of persistent fetal circulation. Elective surgical repair was performed in six infants immediately after cesarean delivery at 35–37 weeks' gestation. In three cases with L/T between 0.21 and 0.24 who recovered with no complications, surgical reduction of the abdominal organs improved arterial blood gases and high-frequency oscillation ventilation (HFOV) was fully effective for respiratory management. In three with L/T between 0.11 and 0.17, extracorporeal membrane oxygenation (ECMO) was required from the 1st to the 12th postoperative day despite HFOV. Although two infants died of combined cardiovascular anomalies and airway bleeding caused by prolonged HFOV, respectively, one infant with minimal L/T survived. Measurement of L/T may help to predict the outcome of fetuses with CDH and to determine the indications for various treatments including immediate operation after cesarean delivery, HFOV, and ECMO. Offprint requests to: S. Kamata     

Clinical usefulness of serum and immunohistochemical markers in patients with stage Ia and Ic ovarian cancer

We compared the preoperative serum tumor marker values and diameters of ovarian tumors between 14 stage Ia ovarian cancer patients with a good prognosis and 14 stage Ic patients with a poor prognosis. The aim was to examine the usability of tumor markers and diameter of ovarian tumors for prognostic diagnosis of clinically advanced phases. In occult neoplastic cells (ONCs), a tumor marker indicative of recurrence and metastasis, the cytokeratin-positive cells in lymph node biopsies, were also compared. In a preoperative comparison of serum tumor markers, CA125 levels in stage Ia and Ic patients were 47.1+/-15.9 (median, 31.9 U/ml) and 370.6+/-146.2 U/ml (median, 135.6 U/ml), respectively (p=0.0457), and CA19-9 levels were 25.5+/-5.5 (median, 20.4 U/ml) and 564.5+/-192.4 U/ml (median, 248.0 U/ml), respectively (p=0.0131). In a comparison of tumor diameters during surgery, diameters of stage Ia and Ic patients were 117.3+/-11.4 (median, 100.0 mm) and 182.0+/-29.2 mm (median, 145.0 mm), respectively (p=0.0457). ONCs were not detected in any stage Ia patients, but detected in 3 (30%) stage Ic patients. In conclusion, clinical progression was evaluated using CA125 and CA19-9 serum markers and tumor diameters in stage Ia and Ic patients, and demonstrated significant differences between stage. ONCs were only detected in the lymph nodes of stage Ic patients.     

Is adrenomedullin involved in the pathophysiology of persistent pulmonary hypertension of the newborn?

Although adrenomedullin (ADM) is a potent vasodilating peptide reported to play a possible role in the mechanisms of fetal lung differentiation and maturation, the ADM blood level in fetuses and in neonates with persistent pulmonary hypertension (PPHN) and pulmonary hypoplasia is not known. Therefore, we examined 15 patients with PPHN: 10 with congenital diaphragmatic hernia, four with congenital cystic adenomatoid malformation of the lung, and one with misalignment of pulmonary vessels with alveolar capillary dysplasia. Eight surgical patients with neonatal conditions such as intestinal atresia served as controls. Blood samples were drawn from the umbilical artery and vein at birth, and arterial blood was drawn from patients with PPHN on the 3rd and 6th days after birth. Plasma levels of ADM were measured by radiometric assay. Plasma levels of ADM in the umbilical artery and vein were elevated in patients with PPHN compared with controls, and in all groups the levels in the umbilical vein were higher than those in the umbilical artery. The arterial levels in patients with poor prognoses were elevated on the 3rd and 6th days after birth compared with those in survivors. These results indicate that ADM may be involved in the pathophysiology of PPHN and in the mechanisms of lung differentiation and/or maturation.     

Intracranial osteosarcoma after radiosurgery--case report

A 56-year-old woman presented with an intracranial osteosarcoma at the site of previous radiosurgery, manifesting as sudden onset of headache and left hemiparesis with aphasia. She had a previous history of stereotactic radiosurgery for an intracranial tumor under a diagnosis of falx meningioma. Computed tomography showed intratumoral and peritumoral hemorrhage at the right parietofrontal region. Gross total resection of the tumor with hematoma was performed. The histological diagnosis was osteosarcoma. Sarcomatous change is a rare complication of radiotherapy. This case illustrates that osteosarcoma may develop years after radiosurgery for benign brain neoplasm.     

Selection criteria for high risk and low risk groups of recurrence and metastasis in patients with primary colorectal cancer

Among 371 patients with primary colorectal cancer, 54 patients suffered from recurrence/metastasis (recurrence group) and 317 survived without recurrence for at least 5 years (non-recurrence group). The clinicopathological characteristics of the 2 groups were compared and occult neoplastic cells (ONCs) in the lymph node sinuses were detected by cytokeratin immunohistochemistry. There were significant differences of the following factors: venous invasion (v-) vs. (v+) for Dukes' A patients (p=0.0315); harvested lymph nodes (LN) or=15 for Dukes' B patients (p=0.0388); (v-) vs. (v+) (p=0.0059), lymphatic invasion (ly-) vs. (ly+) (p=0.0435) for Dukes' A and B patients combined; D>n vs. D=n (p=0.0033), depth of tumor invasion or=se/a2 (p=0.0329) for Dukes' C patients. When the detection of >or=3 ONCs was defined as positive, the sensitivity, specificity, PPV, and NPV were respectively 77%, 100%, 100% and 71% in Dukes' B patients, as well as 75%, 72%, 73% and 74% in Dukes' C patients. The high-risk groups for recurrence/metastasis were identified by the following criteria: (v+) and (ly+), or=se/a2, and ONCs (+) of those with >or=2 factors for Dukes' C patients (selection rate; approximately 21.2-37.5%). These factors seem to be appropriate for separating patients into high-risk and low-risk groups of colorectal cancer recurrence/metastasis.     

Cyanamide-Associated Alcoholic Liver Disease: A Sequential Histological Evaluation

This is the first study that we are aware of that followed the his-topathological progression of the liver disease that was caused by the combination of both chronic alcohol use and cyanamide, an an-tidipsotropic agent. Two sequential liver biopsy specimens were obtained on 29 alcoholics who relapsed with varying histories of cyan-amide treatment. Cyanamide induced ground-glass inclusions (GGIs) in the hepatocytes. Two groups were identified, depending on whether GGIs proliferated or regressed, which was, in turn, found contingent on the duration of cyanamide treatment and the drug-free period. Group 1 included 14 cases in which GGIs either emerged only in the second biopsy specimen or else were increased in the second specimen as compared in the initial specimen. Group 2 composed of 15 cases in which GGIs were either not observed in either specimen or decreased in the second specimen as compared in the initial specimen. Acidophilic bodies were sequentially increased in five cases (36%) of group 1 and in none of group 2. The severity of portal inflammation worsened in 10 cases (71%) of group 1 but in 2 cases (13%) of group 2, although the changes in fibrotic process did not differ between two groups. These differences could not be explained on the basis of the daily ethanol consumption and the length of relapses of the two groups. Thus, when cyanamide-treated alcoholics relapsed, the combined effect of cyanamide and alcohol produced the development of acidophilic bodies and portal inflammation along with the emergence of GGIs.     

Successful laparoscopic cholecystectomy in a child with upper transverse scarring: Report of a case

(Received for publication on Jan. 23, 1997; accepted on Sept. 2, 1997)