Novel biallelic SZT2 mutations in 3 cases of early-onset epileptic encephalopathy.

The seizure threshold 2 (SZT2) gene encodes a large, highly conserved protein that is associated with epileptogenesis. In mice, Szt2 is abundantly expressed in the central nervous system. Recently, biallelic SZT2 mutations were found in 7 patients (from 5 families) presenting with epileptic encephalopathy with dysmorphic features and/or non-syndromic intellectual disabilities. In this study, we identified by whole-exome sequencing compound heterozygous SZT2 mutations in 3 patients with early-onset epileptic encephalopathies. Six novel SZT2 mutations were found, including 3 truncating, 1 splice site and 2 missense mutations. The splice-site mutation resulted in skipping of exon 20 and was associated with a premature stop codon. All individuals presented with seizures, severe developmental delay and intellectual disabilities with high variability. Brain MRIs revealed a characteristic thick and short corpus callosum or a persistent cavum septum pellucidum in each of the 2 cases. Interestingly, in the third case, born to consanguineous parents, had unexpected compound heterozygous missense mutations. She showed microcephaly despite the other case and previous ones presenting with macrocephaly, suggesting that SZT2 mutations might affect head size.

Expression of membrane-type matrix metalloproteinase-1 in human pancreatic adenocarcinomas.

The expression of a new type of matrix metalloproteinase, membrane-type matrix metalloproteinase-1 (MT-MMP-1), was examined in 24 cases of primary pancreatic adenocarcinomas and 9 cases of secondary liver tumors derived from pancreatic adenocarcinomas, using a non-radioactive in situ hybridization and immunohistochemical methods. Out of 24 cases of primary pancreatic adenocarcinomas, 18 showed positive expression of MT-MMP-1 transcripts in cancer cells and 20 of 24 showed positive expression in the tumor stromal cells. The immunoreactivity of the gene products for MT-MMP-1 was demonstrated to be almost the same, as shown by in situ hybridization in these 24 cases. In particular, both the staining intensity for MT-MMP-1 transcripts and the immunoreactivity of the gene products in the tumor stromal cells of mucinous cystadenocarcinomas were significantly weaker than those of common-type ductal adenocarcinomas among the 24 cases. All of the 9 cases of secondary liver tumors derived from pancreatic adenocarcinomas showed positive expression for MT-MMP- transcripts but less immunoreactivity for the gene products. These results suggest that MT-MMP-1 is transcribed and translated in both cancer cells and the tumor stromal cells in human pancreatic adenocarcinomas. Furthermore, considering that common-type ductal adenocarcinoma of the pancreas usually shows a strong desmoplastic reaction, while mucinous cystadenocarcinoma typically does not, MT-MMP-1 expressed in the tumor stromal cells of common-type adenocarcinomas may be involved in processes leading to the desmoplastic reaction.

Allogeneic peripheral blood stem cell transplantation for treatment of induction failure in a patient with Philadelphia chromosome-positive acute lymphoblastic leukemia.

We report a successful case of allogeneic peripheral blood stem cell transplantation (PBSCT) for the treatment of primary induction failure in acute lymphoblastic leukemia (ALL). The patient was a 46-year-old male with Ph-positive ALL and failed to achieve complete remission (CR). His HLA genotypically identical brother refused to donate bone marrow. Instead, PBSCs were collected by apheresis from the brother after administration of G-CSF at a dose of 10 micrograms/kg. For allogeneic PBSCT, the patient was conditioned with marrow ablative chemotherapy and received the PBSC harvest containing 7.8 x 10(4)/kg of CFU-GM, 1.8 x 10(6)/kg of CD34-positive cells and 2.7 x 10(8)/kg of T lymphocytes. After transplant, the neutrophil count exceeded 0.5 x 10(9)/l on day 16 and the platelet count exceeded 20 x 10(9)/l on day 22. CR was confirmed with tri-lineage engraftment in bone marrow samples on days 28 and 49; disappearance of the Ph-chromosome was documented. Sustained engraftment was also confirmed cytogenetically using a variable number of tandem repeat (VNTR) markers. Acute graft-vs.-host disease did not develop with conventional prophylaxis of methotrexate and cyclosporine. However, on day 85 the patient developed leukemia relapse and died on day 97. This clinical trial suggests that allogeneic PBSCT may be an alternative to allogeneic bone marrow transplantation in some limited situations.

Serum succinyltrialanine p-nitroanilide-hydrolytic activity in workers occupationally exposed to lead.

The relationship between blood lead concentration and serum succinyltrialanine p-nitroanilide-hydrolytic (STN) activity was investigated in 74 workers occupationally exposed to lead and in 28 non-exposed workers. Exposure to lead was observed during transfer-printing processes in which paints or paint powders containing 20-50% lead were used. The mean lead concentration was 0.49 mg/m3 (0.12-1.43 mg/m3) in the working environment and 1.42 mg/m3 (0.21-4.34 mg/m3) in workers involved in printing processes. Serum STN activity became lower with increasing concentrations of blood lead (PbB) in non-exposed workers (controls). In lead-exposed workers, the activity decreased with increasing concentrations of PbB of 2.0 mumol/l and more, but increased when less than 1.9 mumol/l. Among lead-exposed workers with high PbB concentrations and/or with hepatic dysfunction, the STN/PbB ratio was distributed above the regression line obtained from the controls. The present investigation suggests that serum STN activity decreases in those highly exposed to lead. Enzymes such as elastase, which hydrolyzes succinyltrialanine p-nitroanilide and additionally is not inhibited by lead, may possibly be induced among lead workers when liver function is impaired.

DNA repair synthesis in primary culture of bovine bile duct epithelial cells induced by chemical agents in relation to bile duct cancer.

Unscheduled DNA synthesis (UDS) was measured autoradiographically in a primary culture of extrahepatic bile duct epithelial cells of Holstein cows following exposure to chemicals known to be capable of developing bile duct cancers in experimental animals, i.e., N-methyl-N'-nitro-N-nitrosoguanidine (MNNG, CAS No. 70-25-7), N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG, CAS No. 4255-77-6), 20-methylcholanthrene (MCA, CAS No. 56-49-5), N-nitrosodimethylamine (DMN, CAS No. 62-75-9) and aflatoxin B1 (AFB1, CAS No. 1162-65-8). MNNG and ENNG induced UDS without addition of S9 mixture. MCA elicited UDS only if S9 mixture was added. Regardless of the presence or absence of S9 mixture, DMN failed to induce UDS. DNA repair by AFB1 was enhanced by the presence of S9 mixture. Therefore, for MNNG, ENNG and high doses of AFB1 activation by the liver is not necessary to exert genotoxic effects and they seem to be capable of direct action on bile duct epithelial cells in the presence of a bilioenteric fistula or anastomosis.

Establishment of the transformants expressing human cytochrome P450 subtypes in HepG2, and their applications on drug metabolism and toxicology.

Transformants with stable expression of a series of human cytochrome P450 (CYP) subtypes in the human hepatic cell line, HepG2, were established. These transformants are designated Hepc/1A1.4, Hepc/1A2.9, Hepc/2A6L.14, Hepc/2B6.68, Hepc/2C8.46, Hepc/2C9.1, Hepc/2C19.12, Hepc/2D6.39, Hepc/2E1.3-8 and Hepc/3A4.2-30, which stably expressed human CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4, respectively. The expression of the CYP subtypes in the transformants was confirmed by both determination of enzyme activities and the reverse transcriptase polymerase chain reaction (RT-PCR) procedure. The apparent K(m) values of the expressed CYP subtypes for their specific substrates were close to those of human liver microsomes. In addition to their CYP activities, these transformants retained glucuronide- and sulfate-conjugating activities. Furthermore, the activities of CYP2C9, CYP2D6 and CYP3A4 were inhibited by their specific inhibitors. The cytotoxicity of acetaminophen (APAP), cyclophosphamide (CPA) and benz[a]anthracene (BA) were analyzed by CYP-expressing transformants. The cytotoxicity depended on the expression of CYP subtypes and increased in a dose-dependent manner. These results show the metabolic activation of APAP, CPA and BA by the specific CYP subtypes expressed in the transformants and demonstrate the usefulness of these transformants for in vitro metabolic and toxicological studies in human liver.

Construction of a sensitive enzyme immunoassay for human fibroblast growth factor 9.

Hybridomas secreting monoclonal antibodies (MAbs) against human fibroblast growth factor 9 (FGF-9) were established using recombinant human (rh) FGF-9 N33 as the immunogen. Among these, MAb 150-59 demonstrated a potent neutralizing activity against FGF-9. It arrested the FGF-9-induced growth of BALB/c 3T3 A31 cells at an equimolar dose of the factor. It also abrogated the in vivo thrombopoietic activity of FGF-9. Mitogenic activity of several other FGF family members such as FGF-1, FGF-2, and FGF-4 was not neutralized by this MAb. A sensitive sandwich enzyme immunoassay for FGF-9 was developed employing MAbs 150-59 and 13-3. The detection limit of this system was 3 pg/well. In this assay system, FGF-1 and FGF-2 were not cross-reactive up to 1 microgram/well. Using this system, the distribution of FGF-9 in rat organs was examined. FGF-9 could be detected only in the extract of rat cerebellum. Also, we detected a high amount of FGF-9 in the culture supernatant of certain cell lines originated from human tumor. These findings suggest that this enzyme immunoassay system may be used to clarify biological meaning of FGF-9.

[Aneurysm of the portal vein associated with aneurysm of the splenic artery]. / Anévrysme de la veine porte associé à un anévrysme de l'artère splénique.

Portal vein aneurysm is very rare. A case of portal vein aneurysm without symptoms of portal hypertension is described. Ultrasonography demonstrated a hypoechoic mass at the isthmic region of the pancreas. Computed tomography demonstrated the vascular origin of the mass. Angiography showed a portal vein aneurysm, measuring 3 cm in diameter, at the junction of portal vein trunk and splenic vein. The association with a splenic artery aneurysm and absence of portal hypertension lead us to suggest that our case is congenital. Non-invasive diagnostic methods are useful to recognize the pathology of portal vein aneurysm and prevent complications especially those related to a possible portal hypertension.

A secondary aortoenteric fistula, successfully treated with proper preoperative diagnosis.

A rare case, presented as a secondary aortoenteric fistula after an abdominal aortic aneurysmectomy with Y-graft replacement 9 months earlier, is reported. The course was clinically very unique, in that the first manifestation of the aorto enteric fistula occurred while the patient had already been hospitalized after the orthopaedic surgical treatment for the pyogenic vertebral spondylitis. After the episode of gastrointestinal bleeding, radiological studies were promptly collected, and with the proper diagnosis, a successful surgical treatment was given under the stable hemodynamic condition, and the patient recovered uneventfully. Retrospectively considered, there are several findings that would suggest that seemingly a secondary aorto enteric fistula could have resulted from an early process of the primary aorto enteric fistula having been under progress without any detectable manifestations before the previous aneurysmectomy. The diagnostic values of computed tomography and the scintigraphy for this rare clinical entity is also underlined.

[Antiplatelet therapy after open heart surgery].

This study evaluated the effects of an antiplatelet drug for the patients after open heart surgery. Dipyridamole (D), aspirin (A), ticlopidine (T) and sarpogrelate hydrochloride (S) were chosen as an antiplatelet drug. The 6 groups were devided into as G 1 with warfarin (W) and D, as G 2 with W alone, as G 3 with A and D, as G 4 with A alone, as G 5 with A and T, and as G 6 with A and S. The indices of anticoagulation and antiplatelet were as follows: TT, APTT, PT, D. Dimer, fibrinogen, TXB2, 6 kPGF1 alpha, beta TG, vWF and platelet aggregation with Grading Curve methods. The results showed that D.Dimer was beyond the normal range in G 1 and G 2. Thromboxane B 2 was increased markedly in G 1 and G 2 and showed under the normal range in G 3 and G 6. In all groups, 6 kPGF1 alpha showed low levels than normal. Activation of platelet causes the increased beta TG which was not suppressed with the antiplatelet drugs administered. The platelet aggregation was not suppressed in G 1 and G 2. Mild to moderate suppression was seen in G 3 and G 4 with no difference between the two group. Moderate to marked suppression was demonstrated in G 5 and G 6. This study demonstrated that dipyridamole had no effects as antiplatelet function. Aspirin showed antiplatelet effects and the combination of aspirin with the another antiplatelet drugs demonstrated the additional effects.

[Two cases of advanced and metastatic breast cancers treated by docetaxel in combination with intra-arterial infusion of adriamycin].

Two cases of advanced and metastatic breast cancers were treated by docetaxel (TXT) in combination with intra-arterial infusion of adriamycin (ADM). Patients received 60 mg/body TXT i.v. and 30 mg/body ADM ia (AT therapy) bi-weekly. Clinical responses were observed in these two patients and the durations of responses were over 20 weeks. Critical toxicities of grade 2 leukopenia and alopecia were observed but grade 4 severe toxicities were not. Thus AT therapy can be easily and safely performed with outpatients. This therapy can improve the response rate and time to progression; therefore phase I or phase I/II clinical trials of AT therapy in Japan are recommended.

An autopsy case of B-cell lymphoma associated with macroglobulinemia (IgM-k).

While he was treated for macroglobulinemia showing symptoms, a 65-year-old man developed an enlargement of the left lateral cervical lymph node. Biopsy specimens of the cervical lymph node revealed large-cell non-Hodgkin's lymphoma. The lymphoma cells had rearrangement in the immunoglobulin heavy chain and kappa light chain genes. There was osteolysis involving right metatarsal bones. And, a tumor appeared on the upper hard palate in the terminal stage. Autopsy specimens showed infiltration of lymphoma cells into multiple organs. Production of monoclonal immunoglobulin M was detected only slightly and focally in the tumors. Plasmacytic differentiation as in Waldenström's macroglobulinemia was not seen in any organs except the tumor on the upper hard palate. From these findings, it is speculated that B-cell lymphoma might have developed in the course of macroglobulinemia.

[Solid and cystic tumor of the pancreas with prominent calcification].

A case of a solid and cystic tumor of the pancreas is presented. The immunohistochemical study of the resected tumor revealed alpha 1-antitrypsin and neuron specific enolase positive malignant cells proliferating pseudopapillary around fibrovascular stroma. Prominent calcification was characteristic in the present case.

[Isolation and culture of bile duct epithelial cells as an useful tool for screening of biliary tract carcinogens].

A method of isolation and culture of extrahepatic bile duct epithelial cells from Holstein cows has been established for detection of carcinogens in relation to bile duct cancer. Cells were isolated by digestion with Dispase followed by scraping of the mucosa. The integrity, the epithelial character and the purity of cells isolated were confirmed by electron microscopy. By the use of this culture induction of unscheduled DNA synthesis by chemicals which are capable of developing bile duct cancers in experimental animals, were assessed autoradiographically with or without addition of S-9 mixture. MNNG and ENNG induced UDS, which was not influenced by S-9 mixture. MCA elicited UDS only if S-9 mixture was added. Regardless of the presence of S-9 mixture, DMN failed to induce UDS but AFB1 induced UDS dose-dependently. The contact routes of the carcinogens with bile duct cells were established and this culture system way be an useful tool for the detection of biliary tract carcinogens.

Anatomical architecture of the brachial plexus in the common hippopotamus (Hippopotamus amphibius) with special reference to the derivation and course of its unique branches.

The anatomy of the brachial plexus in the common hippopotamus (Hippopotamus amphibius), which has not been previously reported, was first examined bilaterally in a newborn hippopotamus. Our observations clarified the following: (1) the brachial plexus comprises the fifth cervical (C5) to first thoracic (T1) nerves. These formed two trunks, C5-C6 and C7-T1; in addition, the axillary artery passed in between C6 and C7, (2) unique branches to the brachialis muscle and those of the lateral cutaneous antebrachii nerves ramified from the median nerve, (3) nerve fibre analysis revealed that these unique nerve branches from the median nerve were closely related and structurally similar to the musculocutaneous (MC) nerve; however, they had changed course from the MC to the median nerve, and (4) this unique branching pattern is likely to be a common morphological feature of the brachial plexus in amphibians, reptiles and certain mammals.

Nerve fibre tracing of branches to the coracobrachialis muscle in a Bornean orangutan (Pongo pygmaeus pygmaeus).

A detailed anatomical analysis of the left brachial plexus, composed of the fourth cervical to first thoracic spinal nerve roots, was performed in an adult male orangutan obtained from the Osaka Museum of Natural History. Although the medial and posterior cords fused into a common trunk, a nerve fibre analysis revealed that the cords were not actually connected. A superficial branch (Rs) running ventral to the musculocutaneous nerve (MC) and a deep branch (Rp) running dorsal to the MC innervated the coracobrachialis muscle, which was also innervated by coracobrachialis branches (Rmc) from the MC. Koizumi (1989: Acta Anat. Nippon, 64, 18) reported that the Rmc and Rs innervated the superficial region of the coracobrachialis muscle corresponding to superficial coracobrachialis muscle in prosimians, whereas the Rp innervated the deep region. However, the detailed innervation of the coracobrachialis muscle in orangutans was not included in Koizumi and Sakai's (1995: J. Anat. 186, 395) report. Our observations in an orangutan did not seem to support generalisation, because the common trunk of the Rp and Rmc appeared to innervate both the superficial and deep regions of the coracobrachialis muscle. Therefore, a nerve fibre analysis of the common trunk was performed as a detailed examination. The analysis confirmed that the Rp in the orangutan only innervated the deep region of the coracobrachialis muscle, similar to the innervation pattern seen in other apes and humans.

Demonstration and maintenance of mucus secretion in cultured human gallbladder epithelial cells.

The method of human gallbladder epithelial cell culture has been developed successfully with active mucus secretory function. Human gallbladder epithelial cells were dissociated by Dispase digestion from the specimens obtained by cholecystectomy for uncomplicated gallbladder stone cases. The dissociated cells formed a monolayer in Eagle's minimum essential medium supplemented with 10% fetal bovine serum within 24 h after the inoculation. These cells were maintained for at least 2 wk without fibroblastic overgrowth. Cultured cells contained periodic acid Schiff-positive material in cellular cytoplasm for 3 d. On transmission electron microscopy these materials were identified as mucous secretory granules. Mucous secretory function was determined by [3H]glucosamine incorporation. Sixty percent of the secreted glycoproteins labeled with [3H]glucosamine was eluted in excluded fractions of Sepharose 4B gel filtration, which were considered to be mucous glycoprotein, because they were found to be resistant to proteoglycan-specific enzymes such as hyaluronidase, chondroitinase ABC, heparitinase, and heparinase. The mucous glycoprotein secretion was maintained for 3 d and found to be inhibited in a dose-dependent manner by monensin (10(-7) to 10(-5) M) which is a known blocker of secretory function.

Liver fibrosis in obese patients with fatty livers.

Liver fibrosis was observed in non-alcoholic, non-diabetic obese patients with fatty liver. The extent of fibrosis was more severe in patients who were obese for a long time and had severe fatty deposition in the liver. Laboratory data were not useful for differentiating between fatty livers with and without liver fibrosis. The results suggested that fatty liver in obese patients should be treated by weight reduction in order to prevent the progression of liver fibrosis.