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.

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.

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.

[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.

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.

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.

Autocrine transformation by fibroblast growth factor 9 (FGF-9) and its possible participation in human oncogenesis.

Transfection of human fibroblast growth factor 9 (FGF-9) cDNA into mouse BALB/c 3T3 clone A31 cells led to morphological transformation of the cells and foci formation 4 weeks later. Isolated transformants had a higher saturation density than parental A31 cells, could grow in soft agar, and secreted FGF-9 into the culture supernatant. The introduction of FGF-9 N33 cDNA, which encodes a truncated protein that has 33 N-terminal amino acids deleted and has the same mitogenic potency as FGF-9, failed to lead to foci formation. Although FGF-9 is a secretory protein, it does not have a typical secretory signal sequence, and the secreted protein retains the full sequence coded in the cDNA except for the initiating methionine. The produced FGF-9 N33 was not secreted and remained within the cell. It is possible that FGF-9 has an uncleavable signal sequence within the first 33 N-terminal amino acids. All of the phenotypes acquired by transformation could be arrested by treatment with a neutralizing anti-human FGF-9 monoclonal antibody (MAb) 150-59. Additionally, transformants formed tumors in nude mice. Injection of MAb 150-59 suppressed tumor formation in nude mice and caused existing tumors to regress. Our results suggest that the cellular transformation mediated by FG F-9 is produced by autocrine stimulation. We have detected FGF-9 production in the human tumor cell lines glioma NMC-G1, from which FGF-9 was originally purified, and stomach carcinoma AZ-521. The growth of NMC-G1 was not affected by MAb 150-59, but that of AZ-521 was arrested by MAb 150-59 in the presence of heparin. Moreover, the growth of the AZ-521 cell tumor in nude mice could be partially arrested by antibody treatment. The possibility of a participation of FGF-9 in the formation of human tumors is suggested.

Exocrine pancreatic function in the early period after pancreatoduodenectomy and effects of preoperative pancreatic duct obstruction.

Exocrine pancreatic function in the early period after pancreatoduodenectomy was investigated. The effects of preoperative pancreatic duct obstruction on exocrine pancreatic function were also investigated. The volume of pancreatic juice and its amylase activity were investigated in 39 patients who underwent pancreatoduodenectomy (including pylorus-preserving pancreatoduodenectomy). The N-benzoyl-L-tyrosyl-p-aminobenzoic acid (BT-PABA) test was performed on 23 of 39 patients about 40 days after pancreatoduodenectomy. The exocrine pancreatic function was inhibited three to eight days after pancreatoduodenectomy (amylase activity: 23,700 +/- 4300 IU/day), and recovered on days 9-15 (48,000 +/- 8400 IU/day) in patients with a normal main pancreatic duct. In patients with pancreatic duct obstruction, the exocrine pancreatic function was almost eliminated (amylase activity: 440 +/- 260 IU/day) and BT-PABA test results were low (45 +/- 17%). In patients with narrowed pancreatic duct, amylase secretion was significantly inhibited even in patients with a normal number of acinar cells. There was a good positive correlation (Spearman's rank correlation coefficient, rs = 0.715, P < 0.01) between amylase secretion and BT-PABA test. Amylase secretion more than 10,000 IU/day is essential for a normal BT-PABA test and normal digestive function. The inhibited digestive function in patients with pancreatic duct obstruction may be due to the decreased number of acinar cells and the inhibition of exocrine pancreatic function.

Electrogastroenterographic examination of 22 patients before and after cholecystectomy.

The object of this study was to define the pattern of gastrointestinal myoelectrical activity before and after cholecystectomy. After surgery, on the first postoperative day, the mean and maximal activities of the gastrointestinal tracts decreased significantly, but there was no significant change in the pattern and the duration of the nonreactive period. A dyskinetic effect and/or weakness of electrical activity was observed in all patients before operation, and the same pattern persisted after operation for one month. This suggests the future onset of the so-called postcholecystectomy syndrome, which may result from the fundamental pathological effect of gallstones.

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.

[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.

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.

Stimulation of thrombopoiesis in mice by fibroblast growth factor 9.

Fibroblast growth factor 9 (FGF-9), a novel member of the FGF family, was found to have thrombopoietic activity in vitro and in vivo. In an in vitro megakaryocyte colony-stimulating factor assay, anti-mouse interleukin-6 (IL-6) monoclonal antibody neutralized FGF-9 activity. This suggests that the activity may be exerted via IL-6 induction. BALB/c mice that received subcutaneous FGF-9 injections of 4 to 100 micrograms/day for 2 weeks showed a dose-dependent transient increase in peripheral platelet counts 10 to 12 days after the first treatment. Histologic studies showed a marked increase in megakaryocytes in bone marrow and extramedullary hematopoiesis in the spleen and the liver. Examination of changes in the DNA content of bone marrow megakaryocytes revealed that the ploidy distribution underwent a marked shift 3 days after FGF-9 injection, with a large increase in the 2N megakaryocyte population. The major modal ploidy shifted from the normal 16N to 2N. The number of megakaryocyte progenitor cells in FGF-9-treated mice increased up to 1.5-fold in the bone marrow and 10-fold in the spleen on day 6. These results indicate that FGF-9 acts on the in vivo proliferation of megakaryocytes.

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.

Platelet-activating factor involvement in the aggravation of acute pancreatitis in rabbits.

Platelet activating factor (PAF) was administered to anesthetized rabbits with cerulein-induced acute pancreatitis to investigate the role of PAF in the development of acute pancreatitis. In acute edematous pancreatitis, induced with cerulein 20 micrograms/kg/h i.v. for 5 h, blood flow in the gastroduodenal and superior mesenteric arteries (GDAF and SMAF) had decreased significantly by 30 min and the serum amylase and lipase levels were significantly increased in the early phase. In the cerulein+PAF group, in which PAF was injected 100 ng/kg/min i.v. for 20 min simultaneously with cerulein, GDAF and SMAF declined significantly to 52 +/- 4 and 47 +/- 3% (p < 0.05), serum amylase and lipase levels rose significantly to 1,110 +/- 150 and 1,370 +/- 190% (p < 0.01) at 300 min, much higher than in the cerulein group. Furthermore, scattered hemorrhages and more marked inflammatory cell infiltration were observed histologically. These findings suggest that PAF has an additive role in the aggravation of acute pancreatitis.

[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.

Relations between lead exposure and peripheral neuromuscular functions of lead-exposed workers--results of tapping test.

In this study, four experiments using the tapping test were conducted to evaluate the possible subclinical effects of lead exposure on the neuromuscular systems of lead workers at some transfer printing factories in Japan. Decreases in the tapping ability appeared coincidentally with higher blood lead levels. The recovery of the tapping ability after 30 sec rest in the groups of 30-45 micrograms/dl and above 45 micrograms/dl PbB was worse than that in the group with less than 29 micrograms/dl PbB. The recovery of the decreased tapping ability after 60 sec rest was better even in the group with 30-45 micrograms/dl PbB. The tapping ability for 0-10 sec at the first tapping test was sustained after 30 or 60 sec rest in the group with the PbB below 29 micrograms/dl; however, the tapping ability at the second and third tapping test decreased in the two groups with the PbB level above 30 micrograms/dl. The decreased finger tapping speed may be functional evidence of low-grade motor neuropathy among the workers with higher levels of lead absorption.

An adult case of bleeding Meckel's diverticulum diagnosed by preoperative angiography.

A 19-year-old woman in her 6th month of pregnancy was admitted to our hospital after passing a massive bloody stool. Romanoscopy and upper gastrointestinal panendoscopy revealed no abnormality, but a superior mesenteric arteriography demonstrated an embryonic artery with the extravasation of contrast medium in the distal end of the ileal artery. After the diagnosis of a bleeding Meckel's diverticulum had been established, an emergency operation was successfully performed. At laparotomy, a Meckel's diverticulum was found, about 70 cm toward the oral side from the ileocecal valve, and part of the ileum, including the diverticulum, was resected. Histological examination indicated that the diverticulum had a normal ileal layer with ectopic gastric mucosa and pancreatic tissue. Furthermore, multiple ulcers were observed in the adjacent ileum. Blood coagulation was observed at the site of one of these ulcers, and this was presumed to be the source of the hemorrhage.