Copy number variations in the amylase gene (AMY2B) in Japanese native dog breeds.

A recent study suggested that increased copy numbers of the AMY2B gene might be a crucial genetic change that occurred during the domestication of dogs. To investigate AMY2B expansion in ancient breeds, which are highly divergent from modern breeds of presumed European origins, we analysed copy numbers in native Japanese dog breeds. Copy numbers in the Akita and Shiba, two ancient breeds in Japan, were higher than those in wolves. However, compared to a group of various modern breeds, Akitas had fewer copy numbers, whereas Shibas exhibited the same level of expansion as modern breeds. Interestingly, average AMY2B copy numbers in the Jomon-Shiba, a unique line of the Shiba that has been bred to maintain their appearance resembling ancestors of native Japanese dogs and that originated in the same region as the Akita, were lower than those in the Shiba. These differences may have arisen from the earlier introduction of rice farming to the region in which the Shiba originated compared to the region in which the Akita and the Jomon-Shiba originated. Thus, our data provide insights into the relationship between the introduction of agriculture and AMY2B expansion in dogs.

Changes in free amino acid concentrations in the blood, brain and muscle of heat-exposed chicks.

An experiment was conducted to analyse the changes in free amino acid concentrations in the blood, brain and muscle of chicks in response to 15 or 30 min exposure to high ambient temperature (HT). Food intake and body weight were not affected, while rectal temperature was significantly increased by short-term HT exposure. Several free amino acid concentrations increased in the blood, brain and muscle even with short-term HT, whereas levels of a few amino acids declined significantly. As well as the nonessential amino acids, essential amino acids also significantly increased with exposure to HT. 3-Methylhistidine, a marker of proteolysis, significantly declined in the muscle of HT chicks, implying a reduction of protein breakdown under HT. These results indicate that alteration of protein metabolism may occur in chicks even with short-term heat exposure.

Important roles of tachykinins in the development of allergic nasal hyperresponsiveness in guinea-pigs.

BACKGROUND: Although it has been suggested that the use of tachykinin receptor antagonists might prove to be an effective treatment for allergic rhinitis (AR), they are not used clinically. Therefore, we decided to examine the effects of tachykinin receptor antagonists on AR symptoms in an appropriate experimental model. OBJECTIVE: To evaluate newly developed tachykinin receptor antagonists in a Japanese cedar pollen-induced AR model and to determine their effect on allergen-induced sneezing, nasal blockage, and nasal hyperresponsiveness (NHR). METHODS: Sensitized guinea-pigs were challenged by forced inhalation of pollen once every week. Sneezing and nasal blockage were observed after pollen challenges. NHR (nasal blockage) to an intranasal application of leukotriene D(4) was assessed 2 days after an antigen challenge. We also evaluated whether intranasal dosing with a tachykinin causes NHR. NK(1) and NK(2) receptor antagonists were administered before an intranasal treatment with antigen or tachykinin. Amounts of tachykinins present in nasal cavity lavage fluid were measured by an enzyme immunoassay. RESULTS: Although an NK(1) and NK(2) receptor dual antagonist showed no effect on pollen-induced sneezing and biphasic nasal blockage, it did completely suppress the development of NHR. Experiments using specific NK(1) or NK(2) receptor antagonists revealed that NK(2) receptor activation was preferentially involved in the development of hyperresponsiveness. Increases in the levels of substance P (SP) and neurokinin A (NKA) in the nasal tissue were noted 20 min-1 h after the challenge. Intranasal instillation of either SP or NKA-induced NHR, which was almost completely inhibited by NK(2) receptor antagonists and partially inhibited by NK(1) receptor antagonists. CONCLUSIONS: SP and NKA, which are released early after the challenge, mediate the development of NHR by preferentially activating NK(2) receptors. Therefore, NK(2) receptor antagonists might prove to be effective treatment of AR.

A new charge-mosaic membrane from a multiblock copolymer.

A charge-mosaic membrane was prepared from a pentablock copolymer of the BABCB type by selectively introducing anion and cation exchange groups into the microseparated phases. The three-layer lamellar structure of the starting pentablock copolymer film was not disturbed by the modifications. The membrane obtained was highly permeable only to sodium chloride in mixed aqueous solutions of sodium chloride and organic species of low molecular weight, such as sucrose. Marked pH-dependent permeabilities were also observed for amino acids.

Molecular identification of a eukaryotic, stretch-activated nonselective cation channel.

Calcium-permeable, stretch-activated nonselective cation (SA Cat) channels mediate cellular responses to mechanical stimuli. However, genes encoding such channels have not been identified in eukaryotes. The yeast MID1 gene product (Mid1) is required for calcium influx in the yeast Saccharomyces cerevisiae. Functional expression of Mid1 in Chinese hamster ovary cells conferred sensitivity to mechanical stress that resulted in increases in both calcium conductance and the concentration of cytosolic free calcium. These increases were dependent on the presence of extracellular calcium and were reduced by gadolinium, a blocker of SA Cat channels. Single-channel analyses with cell-attached patches revealed that Mid1 acts as a calcium-permeable, cation-selective stretch-activated channel with a conductance of 32 picosiemens at 150 millimolar cesium chloride in the pipette. Thus, Mid1 appears to be a eukaryotic, SA Cat channel.

Phospho-regulation of human protein kinase Aurora-A: analysis using anti-phospho-Thr288 monoclonal antibodies.

Mammalian Aurora-A is related to a serine/threonine protein kinase that was originally identified by its close homology with Saccharomyces cerevisiae Ipl1p and Drosophila melanogaster aurora that are key regulators in the orchestration of mitotic events. The protein level of Aurora-A, its peak kinase activity during mitosis, and its activation have been attributed to phosphorylation. Here we show that this enzyme is an arginine-directed kinase and define its substrate specificity. We also found that Thr288 within the activation loop is a critical residue for activating phosphorylation events in vitro and that it is spatiotemporally restricted to a brief window at mitosis on duplicated centrosomes and on spindle microtubules proximal to the poles in vivo. Immunodepletion assays indicated that an upstream kinase(s) of Aurora-A might exist in mammalian cells in addition to autophosphorylation. Furthermore, human activated Aurora-A forms complexes with the negative regulator protein serine/threonine phosphatase type 1 (PP1) that was negatively phosphorylated on Thr320. Interestingly, phospho-specific Aurora-A monoclonal antibodies restrain Aurora-A kinase activity in vitro, providing further therapeutic avenues to explore.

Hematopoietic stem cell transplantation for 30 patients with primary immunodeficiency diseases: 20 years experience of a single team.

We retrospectively analyzed our results of 30 patients with three distinctive primary immunodeficiency diseases (PIDs)--severe combined immunodeficiency (SCID, n = 11), Wiskott-Aldrich syndrome (WAS, n = 11) and X-linked hyper-immunoglobulin M (IgM) syndrome (XHIM, n = 8)--who underwent hematopoietic SCT (HSCT) during the past 20 years. Until 1995, all donors were HLA-haploidentical relatives with T-cell depletion (TCD) (n = 8). Since 1996, the donors have been HLA-matched related donors (MRD) (n = 8), unrelated BM (UR-BM) (n = 7) and unrelated cord blood (UR-CB) (n = 7). Twenty-seven of 30 patients had various pre-existing infections with or without organ damages before HSCT. Conditioning regimen and GVHD prophylaxis were determined according to disease, donor and pretransplant status. Although one of eight patients transplanted with TCD is alive with full engraftment, the other seven died. On the other hand, 18 of 22 patients transplanted without TCD are alive and well, including six of eight transplanted from MRD, seven of seven from UR-BM and five of seven from UR-CB. All 19 survivors did not require Ig supplementation after HSCT. These results indicate that UR-CBT as well as UR-BMT provides good results for PID comparable to MRD-SCT, and that early diagnosis, HSCT at early stage, careful supportive therapy and monitoring for various pathogens are important for the successful HSCT.

Topography Influences Adherent Cell Regulation of Osteoclastogenesis.

The importance of osteoclast-mediated bone resorption in the process of osseointegration has not been widely considered. In this study, cell culture was used to investigate the hypothesis that the function of implant-adherent bone marrow stromal cells (BMSCs) in osteoclastogenesis is influenced by surface topography. BMSCs isolated from femur and tibia of Sprague-Dawley rats were seeded onto 3 types of titanium surfaces (smooth, micro, and nano) and a control surface (tissue culture plastic) with or without osteogenic supplements. After 3 to 14 d, conditioned medium (CM) was collected. Subsequently, rat bone marrow-derived macrophages (BMMs) were cultured in media supplemented with soluble receptor activator of NF-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) as well as BMSC CM from each of the 4 surfaces. Gene expression levels of soluble RANKL, osteoprotegerin, tumor necrosis factor α, and M-CSF in cultured BMSCs at different time points were measured by real-time polymerase chain reaction. The number of differentiated osteoclastic cells was determined after tartrate-resistant acid phosphatase staining. Analysis of variance and t test were used for statistical analysis. The expression of prominent osteoclast-promoting factors tumor necrosis factor α and M-CSF was increased by BMSCs cultured on both micro- and nanoscale titanium topographies (P < 0.01). BMSC CM contained a heat-labile factor that increased BMMs osteoclastogenesis. CM from both micro- and nanoscale surface-adherent BMSCs increased the osteoclast number (P < 0.01). Difference in surface topography altered BMSC phenotype and influenced BMM osteoclastogenesis. Local signaling by implant-adherent cells at the implant-bone interface may indirectly control osteoclastogenesis and bone accrual around endosseous implants.

Accumulation of high levels of the p53 and p130 growth-suppressing proteins in cell lines stably over-expressing cyclin-dependent kinase 6 (cdk6).

Cyclin-dependent kinase 6(cdk6) is present in randomly proliferating cultures of 3T3 cells but has little detectable enzymatic activity. Significant activity is detected only during a short period in early G1 phase. To examine the possible functions of cdk6 in 3T3 cells, lines stably over-expressing cdk6 were constructed and compared to normal 3T3 cells or cell lines with reduced cdk6 levels due to expression of a dominant-negative form of the protein. Over-expression of cdk6 in cells, which led to high levels of activity even in proliferating cultures, had dramatic effects. Cell lines stably over-expressing wild-type cdk6 had a markedly reduced growth rate compared to parental 3T3 cells or lines expressing a dominant-negative form of cdk6. They also over-produced the p53 and p130 proteins and had increased sensitivity to UV-irradiation. Irradiation resulted in accumulation of the Bax protein and rapid cell death. Levels of p53 and p130 proteins were down-regulated and the growth rate of the cells was increased by introduction of the dominant-negative form of cdk6 into cells over-expressing cdk6, indicating that cdk6 is involved in the overproduction of p53 and p130. The results suggest that cdk6, through regulation of growth-suppressing molecules, may play a role in halting cellular growth when proliferation is inappropriate.

A case of gastrointestinal stromal tumour of the ampulla of Vater.

Gastrointestinal stromal tumour rarely develops in the duodenal ampulla region. We report here a case of gastrointestinal stromal tumour of the ampulla of Vater found in a 44-year-old Japanese man presenting with biliary obstruction. He died of hepatic failure with diffuse liver metastasis. The postmortem examination showed a large Borrman type III-like tumour in the duodenal ampullary region with direct invasion of the pancreas and extrahepatic bile duct as well as metastases to the liver and regional lymph nodes. The duct orifice was located at the centre of the tumour. Microscopically, the tumour consisted of anaplastic spindle cells with high mitotic activity (90 mitoses per 50 high-power fields). Immunohistochemically, the spindle cells were positive for KIT and CD34. The final diagnosis was high-grade malignant gastrointestinal stromal tumour of the ampulla of Vater. Considering the recent advances in the diagnosis and treatment of gastrointestinal stromal tumour, this neoplasm should be included in the differential diagnosis of the tumours appearing in the duodenal ampulla region.

Cloning and expression of the epsilon 4 subunit of the NMDA receptor channel.

The primary structure of a novel subunit of the mouse NMDA (N-methyl-D-aspartate) receptor channel, designated epsilon 4, has been revealed by cloning and sequencing the cDNA. The epsilon 4 subunit shares high amino acid sequence identity with the epsilon 1, epsilon 2 and epsilon 3 subunits of the mouse NMDA receptor channel, thus constituting the epsilon subfamily of the glutamate receptor channel. Expression from cloned cDNAs of the epsilon 4 subunit together with the zeta 1 subunit in Xenopus oocytes yields functional NMDA receptor channels. The epsilon 4/zeta 1 heteromeric channel exhibits high apparent affinities for agonists and low sensitivities to competitive antagonists. The epsilon 4 subunit is thus distinct in functional properties from the epsilon 1, epsilon 2 and epsilon 3 subunits, and contributes further diversity of the NMDA receptor channel.

Evaluation of portosystemic collaterals by SPECT imaging after endoscopic variceal sclerotherapy: usefulness for predicting recurrence.

UNLABELLED: Bleeding from esophageal varices is a major cause of morbidity and mortality in cirrhotic patients. Identification of patients at high risk for bleeding is particularly important. The aim of this study was to determine whether detection of portosystemic collaterals by SPECT could predict the outcome of endoscopic injection sclerotherapy of esophageal varices and be useful for selecting appropriate therapy. METHODS: Sixty-two patients with liver cirrhosis who were considered at high risk of bleeding were treated with endoscopic injection sclerotherapy. Endoscopy was performed every 3 mo after therapy or until bleeding occurred. Before and within 2 wk after therapy, tomographic images of intra-abdominal blood pool were constructed by SPECT. RESULTS: Before therapy, the following portosystemic collateral routes were observed: coronary veins in 53 (85.5%) of 62 patients, short gastric veins in 8 patients (12.9%), splenorenal shunts in 10 patients (16.1%), and paraumbilical veins in 6 patients (9.7%). Patients positive for imaging of coronary veins were divided into 3 groups on the basis of changes in images after therapy: complete responders (n = 17), whose coronary vein images disappeared completely; partial responders (n = 18), whose images became smaller; and nonresponders (n = 18), whose images did not change significantly before or after therapy. The rates of recurrence after endoscopic injection sclerotherapy until 6 mo in complete responders (4/17, 23.5%) and partial responders (7/18, 38.9%) were significantly less (P < 0.05) than that in nonresponders (11/13, 84.6%). The rate of recurrence of esophageal varices until 6 mo in nonresponders treated with additional submucosal injection sclerotherapy (1/5, 20.0%) was significantly less (P < 0.05) than that in nonresponders without additional submucosal injection sclerotherapy (11/13, 84.6%). CONCLUSION: Abdominal blood-pool SPECT, a noninvasive method, is useful for evaluating the therapeutic effectiveness of endoscopic sclerotherapy, for predicting the recurrence of varices, and for selecting appropriate management after sclerotherapy.

EBV infection induced transformation of benign T lymphoproliferative state in patient with chronic active EBV infection into malignant lymphoma: implication of EBV infection as additive oncogenic factor in tumorigenesis.

An 11-year-old girl with chronic EBV (Epstein-Barr virus) infection, who later developed malignant lymphoma in the lung, is reported. She had an increased number of V alpha2, V beta8, CD3, CD4, and HLADR positive activated lymphocytes (20-30% of total lymphocytes) in peripheral blood. One year later, she developed lymphoma in the lung, which was V alpha2, V beta8, CD3, CD4, HLADR and IL2Rbeta positive. At that time, the population in the peripheral blood increased up to 40%, but there was no evidence of lymphoma in the bone marrow. In situ hybridization revealed lymphoma cells were EBER-1 positive but gp350/220 and LMP mRNA negative. The EBV genome was detected in the tumor, but not in the peripheral T cells. Clonal analysis of the lymphoma cells revealed monoclonal rearrangement of the TcRbeta and gamma gene, however, investigation of the terminal repeat of EBV gene did not show the monoclonal pattern. These results indicate that infection of EBV into clonally activated T cells was related with transformation from benign lymphoproliferative disease to malignant lymphoma in this patient.

Effects of estrogen and tamoxifen on the MAP kinase cascade in experimental rat breast cancer.

The mitogen-activated protein kinase (MAPK) cascade, which includes MAPK, MAP kinase kinase (MAPKK) and Raf-l, is involved in the signal transduction of growth factor receptors. We found that the MAPK and Raf-l proteins are increased in human breast cancer. Activated MAPKK was also observed. We then investigated whether the MAPK cascade is activated when 7,12-dimethylbenz(a) anthracene (DMBA)-induced rat mammary cancer is treated with 17 beta-estradiol (E-2). Ovariectomy suppressed MAPK expression in tumors, and E-2 administration induced the activation of MAPK in ovariectomized rats. We also investigated the effects of tamoxifen (TAM) on proliferation and the MAPK cascade in DMBA-induced rat mammary cancers. Although tumor size was reduced significantly by TAM, the expression of the MAPK and Raf-l proteins did not decrease. Additionally, MAPK and Raf-l protein expression increased in tumors of ovariectomized rats given TAM, despite a reduction in the size of the tumors. These results suggest that the activated MAPK cascade is important in human breast cancer, and is an important mechanism in the estrogen-dependent growth of DMBA-induced rat mammary cancer. TAM shows E-2-antagonistic effects on tumor proliferation, and E-2-agonistic effects on the MAPK cascade.

Comparative studies using a rigid thoracoscope and fiberoptic bronchoscope to treat spontaneous pneumothorax.

Prior to 1978, the conventional treatment of pneumothorax generally consisted of conservative therapies such as rest, needle puncture, or thoracic cavity drainage; however, when conservative therapies were ineffective or relapse occurred therapeutic approach, 34 percent (11/32) of our patients required a thoracotomy. Consequently, in 1981, we began to use alternative therapies to reduce the need for thoracotomy. In this report, we describe the results of using a rigid thoracoscope and fiberoptic bronchoscope for the treatment of spontaneous pneumothorax. Initially, we treated 31 of 79 patients with a rigid thoracoscope and electrocoagulation therapy. Therapy was effective in 17 (55 percent) of the patients. Only 13 (16 percent) of the 79 patients required a thoracotomy, which represents a 50 percent reduction in incidence. Of the 14 cases in which therapy was ineffective, the major cause of failure was our inability to view a broad thoracic area and treat all blebs with a rigid thoracoscope. Subsequently, we developed a method using a fiberoptic bronchoscope (FB), which allows an unrestricted view of the thoracic area, in combination with electrocoagulation and fibrinogen or thrombin solution (or both) for the treatment of spontaneous pneumothorax. We treated 19 of 39 patients with the FB method. Treatment was effective in 15 (80 percent) of the patients. Only 4 (10 percent) of the 39 patients required a thoracotomy, which is a reduction of over 60 percent in our original incidence.

A case of unilateral diaphragmatic eventration treated by plication with thoracoscopic surgery.

A 56-year-old woman underwent plication with U-stitches by thoracoscopic surgery for left diaphragmatic eventration. Marked improvement in left lung expansion, normalization of the position of the left diaphragm on chest radiograph, and improvement of pulmonary function and dyspnea on exertion have been maintained for 2 years. Plication for diaphragmatic eventration should be performed with minimally invasive surgery.

Gene structure and chromosomal localization of the mouse NMDA receptor channel subunits.

Multiple espilon subunits are major determinants of the diversity of the N-methyl-D-aspartate (NMDA) receptor channel. The four epsilon subunit mRNAs exhibit distinct expression patterns in the brain. In an attempt to elucidate the molecular basis of selective and characteristic expression of the NMDA receptor channel subunits, we have isolated the gene encoding the mouse NMDA receptor epsilon 3 subunit and have determined its structural organization. The epsilon 3 subunit gene spans 17.5 kb and consists of 14 exons. The major transcription start site is 439 bp upstream of the ATG initiation codon as determined by primer extension and S1 nucleas protection analyses. Two polyadenylation sites are 397 (or 398) and 402 bp downstream of the termination codon. The 5'-flanking region of the epsilon 3 subunit gene contains GC-rich segments including consensus sequences for binding of the transcription factors Spl and EGR-1. The murine chromosomal locations of the five NMDA receptor channel subunits, the epsilon 1 (Grin2a), epsilon 2 (Grin2b), epsilon 3 (Grin2c), epsilon 4 (Grin2d) and zeta 1 (Grinl) subunits, were determined using an interspecific backcross mapping panel derived from crosses of [(C57BL/6JxM. spretus) F1xC57BL/6J] mice. Each of these genes mapped to a single chromosome location. The mapping results assigned the five loci to five different mouse autosomes, indicating that they have become well dispersed among mouse chromosomes.

Purification and characterization of two Kunitz family subtilisin inhibitors from seeds of Canavalia lineata.

Two subtilisin inhibitors (CLSI-II and -III) were purified from seeds of Canavalia lineata by extraction with water, ammonium sulfate precipitation, and chromatographies on DEAE-Toyopearl and hydroxyapatite. The two inhibitors have the same molecular weight of about 22,000, and quite similar amino acid compositions. They contain five half-cystine residues and tend to dimerize through an intermolecular disulfide bridge due to the presence of a single cysteine residue. CLSI-III only inhibited subtilisin-type serine proteases, while CLSI-II showed a wider inhibitory specificity. Though the two inhibitors have almost identical thermal labilities, CLSI-II is more stable as to extreme pH than CLSI-III. They are considered to be Kunitz type inhibitors on the basis of several properties.

Pulmonary miliary tuberculosis and T-cell abnormalities in a severe combined immunodeficient patient reconstituted with haploidentical bone marrow transplantation.

We report the development of miliary tuberculosis in a 7-year-old boy with severe combined immunodeficiency (SCID), whose immune system had been only partially reconstituted by haploidentical bone marrow transplantation. Although alpha beta and gamma delta T cells were of donor origin, alpha beta T cells in this patient showed defective interleukin-2 (IL-2) production, impaired IL-2 responsiveness and decreased cytolytic activity. However, gamma delta T cells could exhibit enough cytolytic activity after incubation with IL-2. Despite the presence of disseminated infection, C-reactive protein (CRP) remained negative. IL-2 therapy aggravated the disseminated tuberculosis though gamma delta T cells were supposed to be activated, and concurrently CRP became positive. These findings suggest that gamma delta T cells have no more than limited immunological roles in mycobacterium tuberculosis infection.