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Gene-targeting technology using mouse embryonic stem (ES) cells has become the "gold standard" for analyzing gene functions and producing disease models. Recently, genetically modified mice with multiple mutations have increasingly been produced to study the interaction between proteins and polygenic diseases. However, introduction of an additional mutation into mice already harboring several mutations by conventional natural crossbreeding is an extremely time- and labor-intensive process. Moreover, to do so in mice with a complex genetic background, several years may be required if the genetic background is to be retained. Establishing ES cells from multiple-mutant mice, or disease-model mice with a complex genetic background, would offer a possible solution. Here, we report the establishment and characterization of novel ES cell lines from a mouse model of Alzheimer's disease (3xTg-AD mouse, Oddo et al. in Neuron 39:409-421, 2003) harboring 3 mutated genes (APPswe, TauP301L, and PS1M146V) and a complex genetic background. Thirty blastocysts were cultured and 15 stable ES cell lines (male: 11; female: 4) obtained. By injecting these ES cells into diploid or tetraploid blastocysts, we generated germline-competent chimeras. Subsequently, we confirmed that F1 mice derived from these animals showed similar biochemical and behavioral characteristics to the original 3xTg-AD mice. Furthermore, we introduced a gene-targeting vector into the ES cells and successfully obtained gene-targeted ES cells, which were then used to generate knockout mice for the targeted gene. These results suggest that the present methodology is effective for introducing an additional mutation into mice already harboring multiple mutated genes and/or a complex genetic background.

To generate mutant mice, embryonic stem (ES) cells are used as a vehicle for introducing mutations. The establishment of ES cells is difficult because it requires specific skills and it is time-consuming. We established a novel ES cell line derived from hybrid mice between C57BL/6 and DBA/2 using a modified method. To collect a large number of preimplantational embryos, we collected embryos at the 8-cell stage and cultured them to blastocysts, whereas the usual procedure of preparing the delayed blastocysts demands technical skills. To eliminate unnecessary female cells at an initial stage of inner cell mass culture, male clones were selected by polymerase chain reaction to detect the mouse Sry gene. The established ES cell line efficiently contributed to the germ-line when injected into 8-cell embryos of ICR mice. This potency was maintained after manipulation throughout gene targeting.

We have replaced the NT-3 gene with Escherichia coli-derived lacZ gene by means of homologous recombination in embryonic stem cells and thus produced null mutant mice. Mice homozygous for this mutation developed to birth, but most of them could not suck well and died within 2 days after birth. The surviving homozygous mutant mice displayed movement disorder similar to ataxia. The expression of lacZ was widely distributed in the target tissues of peripheral nerves, spinal motor neurons, lumbar dorsal root ganglia and trigeminal ganglia during the prenatal periods. A neuroanatomical examination revealed that there was marked cell reduction present in trigeminal and lumbar dorsal root ganglia in the developing homozygous mutant mice. In these tissues, the expression of trkC, a high-affinity receptor for NT-3, was markedly reduced. In contrast, we did not find any morphological abnormalities, significant cell loss or decreased levels of trkC expression in the motor neurons present in the ventral horn of the spinal cord. These results indicated that the absence of the NT-3 gene leads to a defect in the sensory nervous system, but it may be complemented by other neurotrophins in the motor nervous system during the development.

Four kinds of retrovirus protease inhibitors (RPI-856 A, B, C and D) were isolated as white powder from the culture filtrate of a soil isolate, Streptomyces sp. AL-322 by column chromatography using Diaion HP-20, Sephadex LH-20, ODS reversed phase HPLC and SP-2SW ion exchange HPLC. The structures of these inhibitors were elucidated by physico-chemical properties, chemical reactions and spectral analyses, as valyl-ADPAA-leucyl-AOPBA-valyl-valyl-aspartic acid (RPI-856 A and B) and valyl-ADPAA-leucyl-AOPBA-valyl-valine (RPI-856 C and D) [ADPAA = 2-amino-2-(3,5-dihydroxyphenyl)acetic acid, AOPBA = 3-amino-2-oxo-4-phenylbutyric acid]. RPI-856 A and B, and RPI-856 C and D were both determined to be diasteromers each other on the asymmetric carbon in AOPBA. These four inhibitors strongly inhibited in vitro HIV-1 protease and HTLV-I protease both derived from recombinant Escherichia coli with IC50 of 10(-7) approximately 10(-8) M.

Vitamin D-24-hydroxylase (CYP24) is one of the enzymes responsible for vitamin D metabolism. CYP24 catalyzes the conversion of 25-hydroxyvitamin D(3) [25(OH)D(3)] to 24,25-dihydroxyvitamin D(3) [24,25(OH)(2)D(3)] in the kidney. CYP24 is also involved in the breakdown of 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)], the active form of vitamin D(3). In this study, we generated transgenic (Tg) rats constitutively expressing CYP24 gene to investigate the biological role of CYP24 in vivo. Surprisingly, the Tg rats showed a significantly low level of plasma 24,25(OH)(2)D(3). Furthermore, the Tg rats developed albuminuria and hyperlipidemia shortly after weaning. The plasma lipid profile revealed that all lipoprotein fractions were elevated in the Tg rats. Also, the Tg rats showed atherosclerotic lesions in the aorta, which greatly progressed with high-fat and high-cholesterol feeding. These unexpected results suggest that CYP24 is involved in functions other than the regulation of vitamin D metabolism.

Four kinds of retrovirus protease inhibitors (RPI-856 A, B, C and D) were isolated as white powder from the culture filtrate of a soil isolate, Streptomyces sp. AL-322 by column chromatography using Diaion HP-20, Sephadex LH-20, ODS reversed phase HPLC and SP-2SW ion exchange HPLC. The structures of these inhibitors were elucidated by physico-chemical properties, chemical reactions and spectral analyses, as valyl-ADPAA-leucyl-AOPBA-valyl-valyl-aspartic acid (RPI-856 A and B) and valyl-ADPAA-leucyl-AOPBA-valyl-valine (RPI-856 C and D) [ADPAA = 2-amino-2-(3,5-dihydroxyphenyl)acetic acid, AOPBA = 3-amino-2-oxo-4-phenylbutyric acid]. RPI-856 A and B, and RPI-856 C and D were both determined to be diasteromers each other on the asymmetric carbon in AOPBA. These four inhibitors strongly inhibited in vitro HIV-1 protease and HTLV-I protease both derived from recombinant Escherichia coli with IC50 of 10(-7) approximately 10(-8) M.

Sigmoid volvulus has often been treated by operative procedures. More recently, however, there has been an increasing number of case reports in which nonoperative reposition has been successfully conducted through the use of an endoscope, especially the colon fiberscope. In this report we present one case of sigmoid volvulus in which nonoperative reposition was achieved using a colon fiberscope. In Japan, to our knowledge, nine studies have so far described endoscopic reposition for this disturbance, including 21 cases in all. This paper presents a discussion of some of those cases for which a detailed description is available together with our case. Compared to four patients who received emergency operation for this disturbance in our clinic, there was no difference with regard to age or duration of affliction; however, the leucocyte count at the time of presentation tended to be higher in the operated group. In addition to evidence of intestinal necrosis and peritoneal irritation, this seems to suggest that evidence of inflammatory signs such as an increased leucocyte count may be helpful in deciding whether to apply nonoperative reposition.

実験的に空腸および回腸切除犬を作製し, 同一犬の切除前と切除後3週間の膵内外分泌動態を比較検討した. SecretinとCCK-PZ混合刺激下での膵液量, 重炭酸濃度はいずれの切除群においても減少し, とくに回腸切除後にはAmylale分泌量も減じた. Glucose, 粗SecretinおよびL. Arginineそれぞれの静脈内投与に対するInsulin分泌反応は小腸切除後にいずれも低下し, L. Arginine刺激下での膵Glucagon分泌反応も低値であつた. これらの傾向は回腸切除後により著明であつた. またL. Arginineに対するGastrin分泌反応はいずれの群においても高分泌反応を示し回腸切除後により著しい傾向を示した. 胆汁分泌量は回腸切除後にとくに著減した. 一方光顕的に膵・肝に変化を認めなかつた.

1. Preoperative arteriography was performed on 45 patients with malignant tumors in the body of the pancreas, which extended to the surroundings far beyond the border of the gland. 2. According to the pattern of tumor infiltration to the splenic artery, these were divided into the normal appearing type, the smooth tapering type, the rough tapering type and the obstructing type, the incidence of which was six, nine, twenty-one and nine, respectively. The former two types belonged to the expansive group, which consisted of cystadenacarcinoma, nonfunctioning islet cell carcinoma, acinar cell carcinoma, sarcoma and carcinoid as well as ductal cell carcinoma. The latter two belonged to the infiltrative group, all of which were ductal cell carcinomas. 3. Almost all of the tumors with increased vascularity belonged to the expansive group, on which effect of chemotherapy might be expectable. 4. On laparotomy, the tumors with the expansive group tended to be associated with liver metastasis, while those with the infiltrative group often showed peritoneal dissemination rather than hematogenous metastasis. 5. Removal of the main tumor could be performed fairly easily for lesions with the normal appearing type, but none of those with the obstructing type was resected. 6. The longest survival was in those with the normal appearing type. In contrast, the poorest prognosis was in those with the obstructing type. In the smooth tapering and the rough tapering types, the prognosis was also poor, but removal of the main tumor in the smooth tapering type resulted in prolongation of survival time. 7. It was found that splenic arteriography can be used as a part of the evaluation of malignant tumors in the body of the pancreas, for which only a few available tools to define its clinical aspects as well as its management have yet been established.

Although many cases of traumatic intramural hematoma in the duodenum in children have been reported, nontraumatic intramural hematoma in the large intestine is extremely rare. Recently, we experienced a case of intramural hamatoma in the sigmoid colon associated with Marfan's syndrome of the incomplete type (forme fruste). This case is described with some comments based on the literature. The patient lacked ophthalmic symptoms and hereditary signs, but had skeletal anomalies. The complication of rupture of a dissecting aortic aneurysm due to median cystic necrosis accompanied by mucoid degeneration occurred after surgery. The patient's condition was thus considered to be Marfan's syndrome of the incomplete type. It is said that lesions in the cardiovascular system in Marfan's syndrome are attributable to abnormalities in the proteins composing the connective tissue or of the system for controlling protein production. The presence of a degenerative condition, Marfan's syndrome, may also have been involved in the formation of the intramural hematoma in the sigmoid colon of this patient.

Small-bowel obstruciton due to gastric bezoar is rare. This case was a 73-year-old gastrectomized woman who was admitted with nausea and abdominal pain and was suspected of having acute pancreatitis. She ate a lot of persimmons several days before the onset of her symptoms and was diagnosed as having gastric bezoar by gastrofiberscopy. Three weeks later severe abdominal pain and distension appeared abruptly. An upper gastrointestinal contrast series demonstrated small-bowel obstruction with a bezoar and emergent operation was performed. A hen-egg-sized bezoar was impacted in the jejunum 30 cm distal from Treitz's ligament. It was removed through enterotomy.Fifteen cases of small-bowel obstruction due to gastric bezoars including this case have been reported during the last 10 years. In all cases conservative treatment failed and laparotomy was necessary. In gastrectomized patients like this case, it is probable that dislodgement of gastric bezoars caused small-bowel obstruction. Therefore, prophylactic measures such as dietary to prevent bezoar formation are important.

Out of breast cancer patients treaked over the past 11 years, clinical investigations were performed on 12 patients with breast cancers complicated by antecedent or concomitant cancers of other organs. The stages of the second cancers (other organ cancers) were relatively more advanced than the accompanying breast cancer and deaths were mostly attributed to these second cancers. A comparative study on the significance of adjunct therapies breast cancer surgery demonstrated that the stages of the second cancers in patients concomitantly given radiation therapy or chemotherapy were more advanced and that the prognosis was aggravated.