A MC motif in silkworm Argonaute 1 is indispensible for translation repression.

Small RNA-mediated gene silencing is a fundamental gene regulatory mechanism, which is conserved in many organisms. Argonaute (Ago) family proteins in the RNA-induced silencing complex (RISC) play crucial roles in RNA interference (RNAi) pathways. In the silkworm Bombyx mori, four Ago proteins have been identified, named as Ago1, Ago2, Ago3 and Siwi. Ago2 participates in double-stranded RNA (dsRNA)-induced RNAi, whereas Ago3 and Siwi are involved in the Piwi-interacting RNA (piRNA) pathway. However, there is no experimental evidence concerning silkworm Ago1 (BmAgo1) in the RNAi mechanism. In the present study, we analysed the function of BmAgo1 in the microRNA (miRNA)-mediated RNAi pathway using tethering and miRNA sensor reporter assays. These results clearly demonstrate that BmAgo1 plays an indispensable role in translation repression in silkworm. Moreover, coimmunoprecipitation data indicated that BmAgo1 interacts with BmDcp2, an orthologue of mRNA-decapping enzyme 2 (Dcp2) protein in the Drosophila processing-bodies (P-bodies). Substitutions of two conserved phenylalanines (F522 and F557) by valines in the MC motif strongly impaired the function of BmAgo1 in translation repression and its localization in P-bodies, suggesting that these two amino acid residues in the MC motif of BmAgo1 are prerequisites for mRNA translation repression in B. mori.

Long-term effects of post-ischaemic oestrogen on brain injury in a rat transient forebrain ischaemia model.

BACKGROUND: The current study was conducted to compare the effects of post-treatment with oestrogen on histological and neurological outcomes after short (7-day) and long (28-day) recovery periods in rats subjected to transient forebrain ischaemia. METHODS: Male Sprague-Dawley rats were randomly assigned to one of five groups: vehicle (7-day recovery period), vehicle (28-day recovery period), oestrogen (17ß-estradiol 200 µg/kg, 7-day), oestrogen (17ß-estradiol 200 µg /kg, 28-day), or sham surgical (n = 8 in each group). After forebrain ischaemia was induced with bilateral carotid artery occlusion and haemorrhagic hypotension (mean arterial pressure = 40 mmHg) for 10 min, the brain was reperfused for 7 or 28 days. Either 17ß-estradiol or vehicle was injected intravenously during the initial 2 min of reperfusion. To evaluate histological damage, the number of intact neurons per 1 mm in the hippocampal CA1 subfield was counted at 7 or 28 days after transient forebrain ischaemia. RESULTS: At 7 days after ischaemia, the number of intact neurons in the hippocampal CA1 subfield was significantly greater in the oestrogen group [57.5 (46.5)/mm: median (interquartile range)] than in the vehicle group [10 (19.5) /mm; P = 0.014]. However, there was no difference between groups at 28 days after ischaemia [vehicle: 11 (20)/mm vs. oestrogen: 6 (11)/mm]. The neurological deficit scores in the oestrogen and vehicle groups were not different from the sham group at any point post-ischaemia. CONCLUSION: The current study indicates that post-ischaemic administration of oestrogen provided short-term but not long-term neuroprotective effects in transient forebrain ischaemia in rats.

Neuroprotective effects of a combination of dexmedetomidine and hypothermia after incomplete cerebral ischemia in rats.

BACKGROUND: Dexmedetomidine and hypothermia are known to reduce neuronal injury following cerebral ischemia. We examined whether a combination of dexmedetomidine and hypothermia reduces brain injury after transient forebrain ischemia in rats to a greater extent than either treatment alone. METHODS: Thirty-eight male Sprague-Dawley rats were anesthetized with fentanyl and nitrous oxide in oxygen. Four groups were tested: group C (saline 1 ml/kg, temporal muscle temperature 37.5 degrees C); group H (saline 1 ml/kg, 35.0 degrees C); group D (dexmedetomidine 100 microg/kg, 37.5 degrees C); and group DH (dexmedetomidine 100 microg/kg, 35.0 degrees C). Dexmedetomidine or saline was administered intraperitoneally 30 min before ischemia. Cerebral ischemia was produced by right carotid artery ligation with hemorrhagic hypotension (mean arterial pressure 40 mmHg) for 20 min. Neurologic outcome was evaluated at 24, 48, and 72 h after ischemia. Histopathology was evaluated in the caudate and hippocampus at 72 h after ischemia. RESULTS: Neurologic outcome was significantly better in the group DH than the group C (P<0.05), whereas it was similar between the group DH and the groups D or H. Survival rate of the hippocampal CA1 neurons was significantly greater in groups D, H, and DH than group C (P<0.05). Histopathologic injury in the caudate section was significantly less in groups H and DH than group C (P<0.05). CONCLUSION: The combination of dexmedetomidine and hypothermia improved short-term neurologic outcome compared with the control group, whereas the combination therapy provided comparable neuroprotection with either of the two therapies alone.

Proposal for a new clinical entity, IgG4-positive multiorgan lymphoproliferative syndrome: analysis of 64 cases of IgG4-related disorders.

BACKGROUND: Mikulicz's disease (MD) has been considered as one manifestation of Sjögren's syndrome (SS). Recently, it has also been considered as an IgG(4)-related disorder. OBJECTIVE: To determine the differences between IgG(4)-related disorders including MD and SS. METHODS: A study was undertaken to investigate patients with MD and IgG(4)-related disorders registered in Japan and to set up provisional criteria for the new clinical entity IgG(4)-positive multiorgan lymphoproliferative syndrome (IgG(4)+MOLPS). The preliminary diagnostic criteria include raised serum levels of IgG(4) (>135 mg/dl) and infiltration of IgG(4)(+) plasma cells in the tissue (IgG(4)+/IgG+ plasma cells >50%) with fibrosis or sclerosis. The clinical features, laboratory data and pathologies of 64 patients with IgG(4)+MOLPS and 31 patients with typical SS were compared. RESULTS: The incidence of xerostomia, xerophthalmia and arthralgia, rheumatoid factor and antinuclear, antiSS-A/Ro and antiSS-B/La antibodies was significantly lower in patients with IgG(4)+MOLPS than in those with typical SS. Allergic rhinitis and autoimmune pancreatitis were significantly more frequent and total IgG, IgG(2), IgG(4) and IgE levels were significantly increased in IgG(4)+MOLPS. Histological specimens from patients with IgG(4)+MOLPS revealed marked IgG(4)+ plasma cell infiltration. Many patients with IgG(4)+MOLPS had lymphocytic follicle formation, but lymphoepithelial lesions were rare. Few IgG(4)+ cells were seen in the tissue of patients with typical SS. Thirty-eight patients with IgG(4)+MOLPS treated with glucocorticoids showed marked clinical improvement. CONCLUSION: Despite similarities in the involved organs, there are considerable clinical and pathological differences between IgG(4)+MOLPS and SS. Based on the clinical features and good response to glucocorticoids, we propose a new clinical entity: IgG(4)+MOLPS.

The combined neuroprotective effects of lidocaine and dexmedetomidine after transient forebrain ischemia in rats.

BACKGROUND: We investigated whether coadministration of lidocaine and dexmedetomidine would reduce brain injury following transient forebrain ischemia in rats to a greater extent than either drug alone. METHODS: Adult male Sprague-Dawley rats were anesthetized with halothane to maintain normocapnia and normoxia. Rats received subcutaneous injection of saline 1 ml/kg, lidocaine 10 mg/kg, dexmedetomidine 3 microg/kg, or lidocaine 10 mg/kg plus dexmedetomidine 3 microg/kg. Thirty minutes after the drug injection, forebrain ischemia was induced by hemorrhagic hypotension and occlusion of the bilateral carotid arteries, and was confirmed by isoelectric EEG. At the end of 10-min ischemia, rats were reperfused. The same dose of drugs was administered 3, 24, and 48 h after ischemia. Neurological examination was done at 1, 2, and 7 days after ischemia. Seven days after ischemia, the brain was stained with hematoxylin and eosin. We counted ischemic cells in the CA1 hippocampal region, striatum, and cerebral cortex. We also measured extracellular glutamate and norepinephrine concentration in hippocampal CA1 in the four groups. RESULTS: As compared with saline-treated rats, rats receiving dexmedetomidine plus lidocaine showed less than neurological deficit scores at 2 and 7 days after ischemia, and had less ischemic cells in the CA1 region. However, administration of dexmedetomidine plus lidocaine did not alter the area under the glutamate concentration curve and norepinephrine concentration during ischemia in the CA1 region, compared with saline-treated rats. CONCLUSIONS: Our results suggest coadministration of lidocaine and dexmedetomidine improves the neurological outcome without alteration of glutamate and norepinephrine concentrations during forebrain ischemia in rats.

[Complete resection of an advanced mediastinal nonseminomatous germ cell tumor with multiple distant metastases after down-staging by chemotherapy].

A mediastinal nonseminomatous germ cell tumor was completely resected after down-staging by chemotherapy despite the presence of multiple distant metastases. A 22-year-old female was admitted for superior vena cava (SVC) syndrome. Her SVC was obstructed by a large anterior mediastinal tumor; she also exhibited distant metastases on a left rib, in the liver, and multiple in the lung. The blood alpha-fetoprotein (AFP) level was extremely elevated to 57,530 ng/ml. Four courses of BEP therapy [cisplatin (CDDP), bleomycin (BLM), etoposide (VP-16)] and a high dose chemotherapy followed by a peripheral blood stem cell transplantation made the tumor become smaller and effected its down-staging. Residual mediastinal tumor with an intravascular tumor in SVC was completely resected. The SVC was reconstructed by an artificial vessel graft. A mediastinal nonseminomatous germ cell tumor, even though it has multiple distant metastases, can achieve down-staging and complete resection by a chemotherapy based on scientific evidence.

Effect of mivazerol, a alpha-agonist, on striatal norepinephrine concentration during transient forebrain ischemia in rats.

BACKGROUND: We have previously reported that mivazerol, a alpha(2)-agonist, possibly provides neuroprotection against transient forebrain ischemia in rats. This study was designed to investigate the ability of mivazerol to attenuate ischemia-induced increase in striatal norepinephrine concentration after transient forebrain ischemia in rats. METHODS: Male Sprague-Dawley rats, anesthetized with halothane, were assigned to one of three groups (n=10 each); control (C, normal saline 1 ml/kg), mivazerol 20 microg/kg (M20), and 40 microg/kg (M40) groups. Monitored variables included temporal muscle temperature (maintained at 37.5+/-0.1 degrees C), electroencephalogram, systolic/diastolic blood pressure, heart rate, arterial blood gases, and blood glucose concentrations. Thirty minutes after subcutaneous drug administration, forebrain ischemia was induced with hemorrhagic hypotension (systolic arterial pressure: 40-50 mmHg) and bilateral carotid artery occlusion for 10 min, and then the brain was reperfused. Norepinephrine concentration in the interstitial fluids in the striatum was analyzed using in vivo microdialysis in combination with high-performance liquid chromatography. RESULTS: Ischemia resulted in a prompt increase in norepinephrine concentrations in the striatum in all groups. However, there were no significant differences in norepinephrine concentrations in the striatum between the three groups at any period. CONCLUSIONS: Our results indicate that mivazerol did not attenuate ischemia-induced increase in striatal norepinephrine concentration. This suggests that the possible neuroprotective property of mivazerol is not related to inhibition of norepinephrine release in the brain.

Anterior pedicle screw fixation for multilevel cervical corpectomy and spinal fusion.

BACKGROUND: Prevention of graft dislodgement in multilevel cervical corpectomy and fusion has been an unresolved problem. Anterior plate fixation has a significant failure rate. External support with a halo-vest is uncomfortable for patients. In the present study, we report a new surgical technique of anterior pedicle screw (APS) fixation for multilevel cervical corpectomy and spinal fusion, and describe the safety and utility of the system. METHOD: After cervical corpectomy, the pedicles on the right side were visualised under oblique fluoroscopy. Guide wires were inserted into the pedicles from the inner wall of the excavated vertebral body until they were hidden in the pedicles. After a fibula autograft was placed, the graft was penetrated in the reverse direction by the guide wires. After drilling and tapping, cannulated screws were inserted into the pedicles through the grafted fibula along the guide wires. FINDINGS: In 9 patients with cervical myelopathy, the surgery was accomplished with a fibula autograft using APS fixation. A total of 22 APSs were inserted, and 21 screws were placed precisely in the pedicles. There were no neurovascular complications. Patients were allowed to ambulate without a halo-vest on the second day after the surgery. Post-operatively, no dislodgement of the grated fibula occurred, and all patients improved neurologically. CONCLUSIONS: The insertion of APSs is feasible and safe. APS fixation enables us to obtain rigid fixation anteriorly, and we propose that APS fixation is an attractive option for multilevel cervical corpectomy and fusion.

Clonality analysis of lymphoproliferative disorders in patients with Sjögren's syndrome.

The aim of this study was to clarify the nature of the clonal lymphocyte infiltration in Sjögren's syndrome (SS) patients associated with lymphoproliferative disorders. We examined B cell clonality in lymphoproliferative tissues from six primary SS patients associated with lymphoproliferative disorders or lymphoma by cloning and sequencing of the gene rearrangement of the immunoglobulin heavy chain complementarity determining region 3 (IgVH-CDR3). Three patients with sequential observation showed progressional clonal expansion with the presence of the same subclone in different tissues during the course of disease. Among them, one patient developed mucosa-associated lymphoid tissue (MALT) lymphoma in glandular parotid. The other three SS patients concomitant with malignant B cells lymphomas showed different clonal expansion of B cells between nodal sites and salivary glands. The cloanality analysis indicated that monoclonal B cell population could spread from one glandular site to another site during the course of SS, suggesting that the malignant clone may arise from the general abnormal microenvironment, not restricted to the glandular tissue, in some SS patients.

Water scarcity hotspots travel downstream due to human interventions in the 20th and 21st century.

Water scarcity is rapidly increasing in many regions. In a novel, multi-model assessment, we examine how human interventions (HI: land use and land cover change, man-made reservoirs and human water use) affected monthly river water availability and water scarcity over the period 1971-2010. Here we show that HI drastically change the critical dimensions of water scarcity, aggravating water scarcity for 8.8% (7.4-16.5%) of the global population but alleviating it for another 8.3% (6.4-15.8%). Positive impacts of HI mostly occur upstream, whereas HI aggravate water scarcity downstream; HI cause water scarcity to travel downstream. Attribution of water scarcity changes to HI components is complex and varies among the hydrological models. Seasonal variation in impacts and dominant HI components is also substantial. A thorough consideration of the spatially and temporally varying interactions among HI components and of uncertainties is therefore crucial for the success of water scarcity adaptation by HI.

Plasma free triiodothyronine response to thyrotropin-releasing hormone to predict the remission of Graves' disease treated with antithyroid drugs.

The responses of both plasma TSH and free T3 (FT3) to TRH were examined in 31 patients with Graves' disease who were euthyroid after treatment with antithyroid drugs, 6 patients with primary hypothyroidism, and 14 control subjects. TSH was measured 0, 15, 30, 60, 90, and 120 min and FT3 was measured 0, 30, 60, 90, 120, 150, and 180 min after TRH injection (500 microgram, iv). The increment in FT3 above the basal level (delta FT3) in normal controls ranged from 1.2-3.7 pmol/L, with a mean +/- SD of 2.2 +/- 0.8 pmol/L. The mean (+/- SD) delta FT3 in patients with primary hypothyroidism was 0.3 +/- 0.2 pmol/L. After the TRH test, antithyroid drugs were stopped in patients with Graves' disease. Nine of 31 Graves' patients relapsed within 6 months after the TRH test. The other 22 patients with Graves' disease were followed while in remission during the observation period of up to 48 months. The mean (+/- SD) delta FT3 were significantly lower in 9 Graves' patients who relapsed than in those who achieved remission (0.5 +/- 0.3 vs. 2.6 +/- 1.1 pmol/L; P less than 0.01). Eight of 9 Graves' patients who relapsed showed lower delta FT3 values than the lowest value (1.1 pmol/L) in 22 Graves' patients in remission. Although the mean increment of TSH above the basal level (delta TSH) was also significantly different between the Graves' patients who relapsed and those in remission (1.4 vs. 12.3 mU/L; P less than 0.01), there was considerable overlap between the 2 groups. These findings suggest that delta FT3 reflects the endocrinological recovery of the pituitary-thyroid axis and is a beneficial indicator for the termination of antithyroid drugs in Graves' disease.

Synthesis of mono- and bifunctional alpha-methylene lactone systems as potential tumor inhibitors.

Synthetic nono- and bifunctional alpha-methylene lactone derivatives including deoxyvernolepin and kihydrodeoxyvernolepin were tested as inhibitors of the growth of CCRF-CEM human lymphoblastic leukemia cells in culture. The range of ID-50 values for compounds 1-7 (ca. 10(-5)-10(-6)M) was roughly comparable to the doses observed earlier in the CCRF-CEM cell system with synthetic alpha-methylene-gamma-butyrolactones. Of significance is that dihydrodeoxyvernolepin and deoxyvernolepin were at least an order of magnitude more active than natural vernolepin.

Suppressive effect of mizoribine on humoral antibody production in DBA/2 mice.

The mode of action of mizoribine (MZR) as a B cell inhibitor was studied using DBA/2 mice. Its in vitro administration significantly delayed the primary response in hemagglutinin production against sheep erythrocytes by suppressing the IgM antibody formation. In vitro plaque-forming cell (PFC) response against both T-dependent and T-independent antigens, such as TNP-SRBC and TNP-Brucella abortus, was dose-dependently suppressed by MZR. Since PFC formation by the T-depleted fraction of splenocytes was likewise suppressed, MZR may inhibit humoral antibody response by directly affecting the B cells (and/or macrophages) as well as by modulating the regulatory T cells. MZR may only act on a certain stage of the cell cycle of B lymphocytes following antigenic stimulation. It may not interfere with the initial antigen recognition or with mature B cells.

Prolongation of the kidney preservation period by simple cold storage up to 72 hours by human atrial natriuretic peptide.

To prolong the preservation period of donor kidneys, we tried to evaluate the effect of human atrial natriuretic peptide (hANP), which has vasodilator, natriuretic, and GFR-increasing effects. Autotransplantations were successful in 6 of 8 hANP-treated cases, but only one of 7 untreated cases (P less than 0.05) was successful. When removed kidneys were perfused prior to preservation, the gravity perfusion time was shorter in hANP-treated kidneys, resulting in rapid cooling. Renal biopsy 1 hr after transplantation revealed normal histology in the hANP-treated kidneys while a remarkable capillary collapse occurred in controls. A hANP-like immunoreactivity contents of biopsied tissue in the hANP-treated kidneys showed a higher value than in the controls, and cyclic GMP contents were also higher in the hANP-treated kidneys. Microangiography showed complete clarity, down to the peripheral vessels, in the hANP-treated kidneys, but not in the controls. Thus, the present study suggests that hANP is useful for prolonging the preservation time of donor kidneys.

Immunosuppressive effect of bredinin on cell-mediated and humoral immune reactions in experimental animals.

Bredinin (BR), an imidazole nucleoside isolated from Eupenicillium brefeldianum was previously reported to prolong kidney allograft survival in dogs. The immunosuppressive effect of BR was studied in experimental animals. In beagles, in vitro responses of lymphocytes stimulated by mitogens or allogeneic cells were suppressed by in vitro BR treatment. BR, given in vivo, also showed an inhibitory action against development of delayed hypersensitivity reaction to tubercle bacilli in guinea pigs or against hemagglutinin production following booster SRBC injection in rabbits. Of note may be the fact that BR was found to have an immunosuppressive potency comparable to that of azathioprine and, in addition, to show a decreased hepatotoxicity compared with the latter.

Induction of ovulation by endothelin-3 in rats.

Endothelins vary in their biological activity. We therefore examined the effects of endothelin-3 (ET-3) on ovulation and secretion of LH, FSH and prolactin in rats in which naturally occurring ovulation was blocked by the administration of sodium pentobarbital (40 mg/kg, i.p.) prior to the critical period (1330 h) on the day of pro-oestrus. ET-3 (10 nmol/kg) was given via the jugular vein under pentobarbital anaesthesia from 1600 to 1800 h on the day of pro-oestrous and induced ovulation in all rats whether given by venous injection or by infusion but the number of ova in rats injected with ET-3 was less than that in normally cycling control rats. Infusion of ET-3 stimulated the secretion of LH but caused a lower than expected rate of secretion of FSH. It would therefore appear that ET-3 causes release of the total amount of LH that is required for induction of ovulation. Our findings strongly suggest that ET-3 has a physiologically significant role in the regulation of anterior pituitary hormone secretion.

The presence of atrial natriuretic peptide in canine cerebrospinal fluid and its possible origin in the brain.

The presence of atrial natriuretic peptide (ANP) in canine cerebrospinal fluid (CSF) was clearly demonstrated and an attempt was made to determine its origin as either the brain or the atrium. The concentration of ANP in canine CSF was 0.78 +/- 0.37 pmol/l (n = 31) and showed no evident correlation with that in plasma (r = 0.12). Physiological doses of human alpha-ANP (alpha-hANP) were continuously infused intravenously into nine dogs, and ANP concentrations in CSF and plasma were examined six to eight times within a 120-min period following this. The ANP level in CSF was not influenced by the systemic administration of alpha-hANP up to 180 min. Only one low molecular weight peak corresponding to alpha-hANP could be obtained from the CSF samples, while both low and high molecular weight peaks were observed for plasma ANP by gel permeation chromatography. In the atrial and hypothalamic tissue extracts the same kinds of peaks were also evident. These results prove the presence of ANP in canine CSF and that it does not come from blood that has seeped across the blood-CSF barriers, but suggest that it may originate from the brain.

Degradation and distribution of brain natriuretic peptide in porcine tissues.

Brain natriuretic peptide (BNP) is a novel peptide that has actions similar to atrial natriuretic peptide (ANP). The present study investigated BNP localization in the heart, ANP and BNP contents in several organs, and ANP and BNP clearance through these organs. In the morphological study, it was shown that porcine BNP-like immunoreactivity was mainly distributed in the granules of the atrium. The content of porcine BNP-like immunoreactivity in the atrium was extremely high, about 100-fold greater than in the ventricle. From the determination of porcine BNP and ANP contents of such organs as the heart, kidney and liver and also plasma, it was shown that porcine BNP concentration was approximately one order of magnitude lower than that of ANP, and clearance rates of ANP and porcine BNP from these organs were similar and not significantly different between the organs. These results suggest that the modes of secretion and degradation of porcine BNP are not the same as those of ANP.

The role of the left ventricle in atrial natriuretic peptide secretion in acute mitral regurgitation.

To examine the role of the ventricle in ANP secretion, the atrial natriuretic peptide (ANP) concentration in the plasma, left atrial tissue and left ventricular tissue were observed in a canine during the course of experimental reversible mitral regurgitation (MR). For reversible MR, a basket catheter was inserted into the left atrium via the pulmonary vein and fixed at the mitral valve. The regurgitation arose when the tip basket wire was extended and disappeared immediately when it was closed. Left atrial pressure (LAP), right atrial pressure (RAP) and pulmonary artery pressure (PAP) increased significantly during MR, and decreased to the normal level after recovery from MR. Plasma ANP concentration showed a reversible change in correlation with LAP and RAP in each canine studied. The ANP level in the atrial and ventricular tissue decreased during MR, and the low levels were maintained after recovery from MR. Both the left arterial and left ventricular tissue ANP levels showed the same changes in acute MR. Therefore, it was suggested that normal ventricles might play a role in ANP secretion in acute MR.

The amino acid sequences of proteinase inhibitors I-A and I-A' from adzuki beans.

Several proteins which strongly inhibit trypsin have been found in adzuki bean seeds. Two of them, designated as adzuki proteinase inhibitors (API) I-A and I-A', were analyzed for their amino acid sequences by conventional methods. Inhibitors I-A and I-A' exhibited strong homology with other Bowman-Birk type proteinase inhibitors from leguminous seeds in spite of belonging to different genera. Inhibitors I-A and I-A' consisted of 78 and 72 amino acid residues and their molecular weights were 9,100 and 8,300, respectively. Inhibitor I-A' lacked the six amino acid residues of the amino terminus of inhibitor I-A and had an asparagine residue in place of the aspartic acid residue at position 40 of inhibitor I-A. The results showed the occurrence of some genetic variants of proteinase inhibitors in adzuki bean seeds. Inhibitor I-A was a double-headed one, and the reactive sites for trypsin were Lys-Ser and Arg-Ser bonds. Therefore, inhibitor I-A' was also assumed to be a double-headed one having Lys-Ser and Arg-Ser bonds as the reactive sites for the enzyme.