Distribution of (125)I-labeled crotamine in mice tissues.

Crotamine is a strong basic polypeptide from Crotalus durissus terrificus (Cdt) venom composed of 42 amino acid residues tightly bound by three disulfide bonds. It causes skeletal muscle spasms leading to spastic paralysis of hind limbs in mice. The objective of this paper was to study the distribution of crotamine injected intraperitoneally (ip) in mice. Crotamine was purified from Cdt venom by gel filtration followed by ion exchange chromatography, using a fast-performance liquid chromatography (FPLC) system. Purified crotamine was irradiated at 2 kGy in order to detoxify. Both native and irradiated proteins were labeled with (125)I using chloramine T method, and separated by gel filtration. Male Swiss mice were injected ip with 0.1 mL (2 x 10(6)cpm/mouse) of (125)I native or irradiated crotamine. At various time intervals, the animals were killed by ether inhalation and blood, spleen, liver, kidneys, brain, lungs, heart, and skeletal muscle were collected in order to determine the radioactivity content. The highest levels of radioactivity were found in the kidneys and the liver, and the lowest in the brain.

New view on crotamine, a small basic polypeptide myotoxin from South American rattlesnake venom.

Crotamine is a toxin from the Crotalus durissus terrificus venom, composed of 42 amino acid residues and three disulfide bridges. It belongs to a toxin family previously called Small Basic Polypeptide Myotoxins (SBPM) whose members are widely distributed through the Crotalus snake venoms. Comparison of SBPM amino acid sequences shows high similarities. Crotamine induces skeletal muscle spasms, leading to spastic paralysis of the hind limbs of mice, by interacting with sodium channels on muscle cells. The crotamine gene with 1.8 kbp is organized into three exons, which are separated by a long phase-1 and short phase-2 introns and mapped to chromosome 2. The three-dimensional structure of crotamine was recently solved and shares a structural topology with other three disulfide bond-containing peptide similar to human beta-defensins and scorpion Na+ channel toxin. Novel biological activities have been reported, such as the capacity to penetrate undifferentiated cells, to localize in the nucleus, and to serve as a marker of actively proliferating living cells.

Effects of 60Co gamma radiation on crotamine

Ionizing radiation can change the molecular structure and affect the biological properties of biomolecules. This has been employed to attenuate animal toxins. Crotamine is a strongly basic polypeptide (pI 10.3) from Crotalus durissus terrificus venom composed of 42 amino acid residues. It induces skeletal muscle spasms leading to a spastic paralysis of hind limbs in mice. The objective of the present study was to carry out a biochemical study and a toxic activity assay on native and irradiated crotamine. Crotamine was purified from C.d. terrificus venom by Sephadex G-100 gel filtration followed by ion-exchange chromatography, and irradiated at 2 mg/ml in 0.15 M NaCl with 2.0 kGy gamma radiation emitted by a 60Co source. The native and irradiated toxins were evaluated in terms of structure and toxic activity (LD50). Irradiation did not change the protein concentration, the electrophoretic profile or the primary structure of the protein although differences were shown by spectroscopic techniques. Gamma radiation reduced crotamine toxicity by 48.3 percent, but did not eliminate it

Effects of (60)Co gamma radiation on crotamine.

Ionizing radiation can change the molecular structure and affect the biological properties of biomolecules. This has been employed to attenuate animal toxins. Crotamine is a strongly basic polypeptide (pI 10.3) from Crotalus durissus terrificus venom composed of 42 amino acid residues. It induces skeletal muscle spasms leading to a spastic paralysis of hind limbs in mice. The objective of the present study was to carry out a biochemical study and a toxic activity assay on native and irradiated crotamine. Crotamine was purified from C.d. terrificus venom by Sephadex G-100 gel filtration followed by ion-exchange chromatography, and irradiated at 2 mg/ml in 0.15 M NaCl with 2.0 kGy gamma radiation emitted by a 60Co source. The native and irradiated toxins were evaluated in terms of structure and toxic activity (LD50). Irradiation did not change the protein concentration, the electrophoretic profile or the primary structure of the protein although differences were shown by spectroscopic techniques. Gamma radiation reduced crotamine toxicity by 48.3%, but did not eliminate it.

The utility of endoscopic ultrasonography and endoscopy in the endoscopic mucosal resection of early gastric cancer.

OBJECTIVE: To clarify the usefulness of endoscopic ultrasonography (EUS) and endoscopy in the endoscopic mucosal resection (EMR) of early gastric cancer. Patients/Methods-EMR was performed in 61 patients with early gastric cancer over the past five years. The accuracy of the assessment of the depth of cancerous invasion was studied in 49 patients who had EUS before EMR. Forty eight patients were treated with endoscopy alone; in these patients, EUS and endoscopic findings correlated with the clinical course. RESULTS: Forty six patients showed no changes in the submucosal layer or deeper structures on EUS. Pathologically these included 37 patients with mucosal cancer and nine with submucosal cancer showing very slight submucosal infiltration. Three patients showed diffuse low echo changes in the submucosal layer on EUS; pathologically, these included two with submucosal cancer and one with mucosal cancer with a peptic ulcer scar within the tumour focus. Of 48 patients receiving endoscopic treatment alone, 45 showed no tumour recurrence or evidence of metastases on EUS and endoscopy. Three cases of recurrence were observed. Two of these patients had a surgical gastrectomy, and one was re-treated endoscopically. In the former cases, the surgical results correlated well with assessment by EUS and endoscopy. In addition, the latter patient who was re-treated endoscopically after evaluation with EUS and endoscopy has so far had no recurrence. CONCLUSION: The combined use of EUS and endoscopy is effective in diagnosing the depth of cancerous invasion in patients undergoing EMR as well as in clarifying changes both within and between anatomic levels during follow up.

[Value of MR cholangiography in the diagnosis of pancreatic diseases compared with endoscopic retrograde cholangiopancreatography].

PURPOSE: To compare magnetic resonance cholangiography (MRCP) with endoscopic retrograde cholangiography (ERCP) in the diagnosis of pancreatic diseases. METHODS: The authors retrospectively reviewed MRCP and ERCP images obtained in 56 patients with pancreatic diseases and 192 controls. Detectability of the pancreatic duct and its branches and diagnostic usefullness were evaluated. RESULTS: MRCP was successful in almost all the patients without ascitic fluid or ileus. Findings of the main pancreatic ducts (i.e., dilation, stenosis and obstruction) were similarly demonstrated by ERCP and MRCP. The degree of ductal narrowing tends to be overestimated with MRCP. CONCLUSION: MRCP appears to be less invasive than ERCP. MRCP could provide sufficient information and play a complementary role in the diagnosis of pancreatic diseases.

Aggressive non-Hodgkin's lymphoma after successful eradication of Helicobacter pylori and regression of gastric lymphoma of mucosa-associated lymphoid tissue.

A 57-year-old woman presented to our clinic with low-grade gastric lymphoma of mucosa-associated lymphoid tissue (stage IE) and Helicobacter pylori infection. She received a 2-week course of omeprazole and clarithromycin, resulting in eradication of H. pylori and histological disappearance of the lymphoma. However, 9 months later (May 1996), multiple mass lesions were found around the pancreas and hepato-duodenal ligament on abdominal computed tomography. Inguinal lymph node biopsy revealed aggressive nodal type B-cell non-Hodgkin's lymphoma, diffuse large cell type. She received chemotherapy with cyclophosphamide, adriamycin, vincristine, and prednisolone, but failed to achieve remission and died in December 1996. There was no evidence of recurrent gastric lymphoma. This case emphasizes the importance of performing follow-up examinations to detect other neoplasms in patients with gastric lymphoma of mucosa-associated lymphoid tissue.

A single-step purification of bothropstoxin-1

Bothrops venoms are complex mixtures of components with a wide range of biological activities. Among these substances, myotoxins have been investigated by several groups. Bothropstoxin-1 (Bthtx-1) is a phospholipase A2-like basic myotoxin from Bothrops jararacussu. The purification of this component involves two chromatographic steps. Although providing a pure material, the association of these two steps is time consuming and a single-step method using high performance chromatography media would be useful. In the present study, we describe a single-step purification method for Bthtx-1. Bothrops jararacussu venom was dissolved in 1 ml buffer. After centrifugation, the supernatant was injected into a Resource-S cation exchange column connected to an FPLC system and eluted with a linear salt gradient. The complete procedure took 20 min, representing a considerable time gain when compared to a previously described method (Homsi-Brandenburgo MI et al. (1988) Toxicon, 26: 615-627). Bthtx-1 purity and identity, assessed by SDS-PAGE and N-terminal sequencing, resulted in a single band with a molecular mass of about 14 kDa and the expected sequence of the first 5 residues, S-L-F-E-L. Although the amount of protein purified after each run is lower than in the previously described method, we believe that this method may be useful for small-scale purifications

A single-step purification of bothropstoxin-1.

Bothrops venoms are complex mixtures of components with a wide range of biological activities. Among these substances, myotoxins have been investigated by several groups. Bothropstoxin-1 (Bthtx-1) is a phospholipase A2-like basic myotoxin from Bothrops jararacussu. The purification of this component involves two chromatographic steps. Although providing a pure material, the association of these two steps is time consuming and a single-step method using high performance chromatography media would be useful. In the present study, we describe a single-step purification method for Bthtx-1. Bothrops jararacussu venom was dissolved in 1 ml buffer. After centrifugation, the supernatant was injected into a Resource-S cation exchange column connected to an FPLC system and eluted with a linear salt gradient. The complete procedure took 20 min, representing a considerable time gain when compared to a previously described method (Homsi-Brandenburgo MI et al. (1988) Toxicon, 26: 615-627). Bthtx-1 purity and identity, assessed by SDS-PAGE and N-terminal sequencing, resulted in a single band with a molecular mass of about 14 kDa and the expected sequence of the first 5 residues, S-L-F-E-L. Although the amount of protein purified after each run is lower than in the previously described method, we believe that this method may be useful for small-scale purifications.

Ultrasonographic angiography in gallbladder diseases.

Ultrasonographic (US) angiography was performed by sonographic examination with injection of carbon dioxide microbubbles through a catheter following conventional angiography in 41 patients with various gallbladder diseases. Three enhancement patterns were found; strong enhancement in the lesion from the periphery to the center was noted in patients with adenocarcinoma and benign polyp (type I), irregular partial enhancement at the margins of the lesion in those with adenosquamous carcinoma (type II), and internal regular enhancement in those with chronic cholecystitis, xanthogranulomatous cholecystitis and adenomyomatosis (type III). Pseudopolypoid lesion such as gallbladder debris showed no enhancement. US angiography may be useful in the differential diagnosis of gallbladder diseases, especially to differentiate the wall thickening type of gallbladder carcinoma from chronic cholecystitis or adenomyomatosis, and the pseudopolypoid lesion and fundal type of adenomyomatosis from benign polyp or polypoid-type carcinoma.

Differential diagnosis of pancreatic diseases with an intraductal ultrasound system.

Forty patients with pancreatic diseases (11 cancer, 1 islet cell tumor, 12 mucin-producing tumor, 1 teratoma, and 15 chronic pancreatitis) were studied in vivo with ERP and were also scanned with an intraductal ultrasound (IDUS) probe at a frequency of 30 MHz inserted into the main pancreatic duct. The usefulness of in vivo IDUS was evaluated by comparison of images with the ERP findings. IDUS was of diagnostic value in 18 of the 40 patients; it distinguished between 4 malignant and 6 benign causes of localized stenosis revealed by ERP, provided parenchymal information in 2 cases with only displacement revealed by ERP (1 islet cell tumor and 1 teratoma), and determined the extent of tumor in 6 cases with main-duct-type mucin-producing tumor. Ten of 11 cancer, 4 of 12 mucin-producing tumor, 1 islet cell tumor, and 11 of 15 chronic pancreatitis (previously scanned in vivo), and 2 islet cell tumor (not scanned in vivo), were resected and scanned in vitro. Fifteen normal pancreases from autopsy subjects were also scanned in vitro. The IDUS images were then compared with corresponding histopathological sections from the 15 normal pancreases and 28 post-operative pancreatic specimens. Differential diagnosis of the pancreatic diseases by echo patterns was possible in all cases except those with intraductal papillary adenocarcinoma and adenoma.

[The utility of endoscopic ultrasonography (EUS) in endoscopic mucosal resection of early gastric cancer].

The utility of EUS was evaluated in 27 patients with early gastric cancer undergoing endoscopic mucosal resection over the past 2 years. Accuracy of the assessment of depth of cancerous invasion was studied in 16 patients undergoing EUS before endoscopic mucosal resection. Patients showing no changes in the submucosal (sm) layer or below on EUS included 15 with mucosal (m) cancer and one with sm cancer showing very slight infiltration. Seven patients with m cancer, a negative stump, and no ulcer in the cancer focus at endoscopic mucosal resection, were followed up for more than 1 year after endoscopic mucosal resection. On EUS, four patients showed Ul-IIs changes resembling benign ulcers, two showed Ul-IIIs changes and two showed no changes in the sm layer or below. All patients were negative for cancer in follow up biopsies. No lymphadenopathy was observed. EUS was effective in diagnosing the depth of cancerous invasion in patients undergoing endoscopic mucosal resection and also in clarifying changes in the sm and deeper layers during follow up.

[Examination of cholangiogram of obstructive jaundice using MRI--compared with PTC and ERCP].

PTC and ERCP are most often used in diagnosis of obstructive jaundice. We studied the possibility of clinical diagnosis using MRI in 33 cases of obstructive jaundice. A clinical diagnosis of malignant tumors could be given in 17 cases out of 20 (85%) using MRI if respiratory standstill was possible. An MRI cholangiogram was particularly effective in describing tearful parting bile ducts and was clearer than PTC in describing negative gallbladders. Choledochal stones could be diagnosed in 58% of cases, which was less than the rate for malignant tumors. MRI is not an invasive examination, can be used in diagnosis of obstructive jaundice, and helps in selecting treatment methods such as PTCD and ERBD.

New technique using intraductal ultrasonography for the diagnosis of diseases of the pancreatobiliary system.

Intraductal ultrasonography (IDUS), a new technique for visualizing arterial structures, operates at an ultrasound frequency of 30 MHz to produce high resolution, cross-sectional images in real time. The purpose of this study was to provide a basis for interpreting IDUS images in vitro. We also attempted to determine the clinical usefulness of the IDUS system in diagnosing pancreatobiliary diseases in vivo. IDUS echograms of both the bile duct (BD) and main pancreatic duct (MPD) from autopsy specimens of 15 patients demonstrated three distinct layers with a fine reticular pattern in the pancreas in vitro. In clinical cases, the MPD and BD of four patients could be scanned by inserting the IDUS catheter via the major papilla without requiring endoscopic sphincterotomy. We hope that IDUS will become routine in scanning the BD and MPD to achieve early and accurate diagnoses of pancreatobiliary diseases.

[Imaging diagnosis of the insulinoma--the clinical usefulness of intraductal ultrasonography of the pancreas and color Doppler sonography].

5 cases of small-sized insulinoma of the pancreas were studied to evaluate the ability of diagnostic imaging techniques to determine tumor location. Techniques used were extracorporeal ultrasonography, endoscopic ultrasonography (EUS), angiography, percutaneous transhepatic portal catheterization and CT. EUS was superior to the other modalities in diagnosing the location of insulinomas. We employed Color Doppler (CD) ultrasonography in one case and obtained color expression in the tumor and a pulsatile wave in the FFT analysis. Intraductal sonography of the pancreas (IDSP) was also performed in this case resulting peripheral echo-lucent, central echogenic mass. From these results, we expect that CD and IDSP will be useful in clinical application for diagnosing various tumors.