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ペインクリニックにおける加療中に,造血器悪性腫瘍が発見された2症例を経験した.[症例1]50歳,女性.左口唇周囲の小水疱と,左下顎部のしびれ・疼痛などを主訴に受診した.この症候はnumb chin syndromeとよばれ,進行した悪性腫瘍の存在を示唆する症候群であった.精査により,バーキット型急性リンパ性白血病と診断されたが第43病日に死亡した.[症例2]74歳,女性.骨粗霧症と胸推圧迫骨折による腰背痛のために加療していたが,初診より7ヵ月を経てから多発性骨髄腫と診断された.化学療法は奏効し,初診後2年を経た現在,疼痛とADLの改善が得られている.未診断のまれな悪性疾患に由来する疼痛にも留意すべきである.

The aim of this study was to estimate genetic correlations between milk yield, somatic cell score (SCS), mastitis, and claw and leg disorders (CLDs) during first lactation in Holstein cows by using a threshold–linear random regression test-day model. We used daily records of milk, fat and protein yields; somatic cell count (SCC); and mastitis and CLD incidences from 46 771 first-lactation Holstein cows in Hokkaido, Japan, that calved between 2000 and 2009. A threshold animal model for binary records (mastitis and CLDs) and linear animal model for yield traits were applied in our multiple trait analysis. For both liabilities and yield traits, additive genetic effects were used as random regression on cubic Legendre polynomials of days on milk. The highest positive genetic correlations between yields and disease incidences (0.36 for milk and mastitis, 0.56 for fat and mastitis, 0.24 for protein and mastitis, 0.32 for milk and CLD, 0.44 for fat and CLD and 0.31 for protein and CLD) were estimated at about the time of peak milk yield (36 to 65 days in milk). Selection focused on early lactation yield may therefore increase the risk of mastitis and CLDs. The positive genetic correlations of SCS with mastitis or CLD incidence imply that selection to reduce SCS in the early stages of lactation would decrease the incidence of both mastitis and CLD.

The objective of this study was to estimate genetic parameters and breeding values for the twinning rate of the first three parities (T1, T2 and T3) and 305-day milk yield in first lactation (MY), using a four-trait threshold-linear animal model in Japanese Holsteins. Data contained 1 323 946 cows calving between 1990 and 2007. Twinning was treated as a binary character: 'single' or 'twin or more'. Reported T1, T2 and T3 were 0.70%, 2.87%, and 3.73%, respectively. Individual 305-day milk yield was computed using a multiple trait prediction for cows with at least eight test-day records. (Co)variance components were estimated via Gibbs sampling for randomly sampled subsets. Posterior means of heritabilities for T1, T2 and T3 were 0.11, 0.16 and 0.14, respectively. Genetic correlations between parities were 0.92 or greater. Genetic correlations of MY with twinning rate were not 'significant' (i.e. their 95% highest probability density intervals contained zeros). Multiple births at different parities were considered as the same genetic trait. The average evaluations of T1 (T2) for sires born before 1991 was 0.48% (2.25%) compared with a mean of 0.76% (3.37%) for sires born after 1992. A recent increase in the reported twinning rate reflects the positive genetic trend for sires in Japanese Holsteins.

Guillain-Barre syndrome, which is a variant of acute inflammatory neuropathy, is associated with anti-GM1 antibodies and causes ataxia. We investigated the effects of IgG anti-GM1 monoclonal antibody (IgG anti-GM1 mAb) on spontaneous muscle action potentials in a rat spinal cord-muscle co-culture system and the localization of IgG anti-GM1 mAb binding in the rat hemi-diaphragm. The frequency of spontaneous muscle action potentials in innervated muscle cells was acutely inhibited by IgG anti-GM1 mAb. When cultures were pretreated with GM2 synthase antisense oligodeoxynucleotide, IgG anti-GM1 mAb failed to inhibit spontaneous muscle action potentials, demonstrating the importance of the GM1 epitope in the action of IgG anti-GM1 mAb. Immunohistochemistry of rat hemi-diaphragm showed that IgG anti-GM1 mAb binding overlapped with neurofilament 200 (NF200) antibodies staining, but not α-bungarotoxin (α-BuTx) staining, demonstrating that IgG anti-GM1 mAb was localized at the presynaptic nerve terminal. IgG anti-GM1 mAb binding overlapped with syntaxin antibody and S-100 antibody in the nerve terminal. After collagenase treatment, IgG anti-GM1 mAb and NF200 antibodies did not show staining, but α-BuTx selectively stained the hemi-diaphragm. IgG anti-GM1 mAb binds to the presynaptic nerve terminal of neuromuscular junctions. Therefore, we suggest that the inhibitory effect of IgG anti-GM1 mAb on spontaneous muscle action potentials is related to the GM1 epitope in presynaptic motor nerve terminals at the NMJs.

It was demonstrated recently that geranylgeraniol (GGO) has potent apoptosis-inducing activity in various lines of tumor cells, including HL-60 cells. In the present study, we found that GGO markedly inhibited the expression of a calcium-binding protein, calreticulin, prior to the induction of apoptosis in HL-60 cells. Furthermore, we also observed a significant decrease in the tyrosine phosphorylation of a 36-kDa protein that is a major tyrosine-phosphorylated protein in HL-60 cells. These findings suggest that decreases in levels of calreticulin and in the tyrosine phosphorylation of the 36-kDa protein might be associated with the induction of apoptosis by GGO in HL-60 cells.

We isolated and characterized almost the entire cDNA encoding BLM hydrolase from rat spleen cDNA libraries. The cDNA encoded a polypeptide composed of 454 amino acids, that had a slightly larger molecular mass than that was previously estimated by SDS-PAGE for purified BLM hydrolase subunit. Amino acid sequence alignments showed that rat BLM hydrolase is homologous to that of rabbit (94% identity of partial amino acid sequence), yeast cysteine protease (39% identity), and the pepC gene products of three bacteria (34-40% identity). In addition, it contained the three regions that are conserved in other cysteine proteases and thought to constitute the catalytic center. These results indicated that rat BLM hydrolase is a member of the papain superfamily of cysteine proteases. Sequencing revealed several putative sites phosphorylated by different types of protein kinases, but no signal sequence, transmembrane domain, N-linked glycosylation site or DNA-binding motif. The yeast homolog is a DNA-binding cysteine protease [Xu et al. (1994) J. Biol. Chem, 269, 21177-21183]. We demonstrated that rat BLM hydrolase also binds the single-stranded form of the Ga14 DNA-binding site oligonucleotide with high affinity compared with that of the double-stranded form. Northern blots revealed that the level of BLM hydrolase mRNA expression was very low in the rat skin, lung, and skeletal muscle. Furthermore, BLM hydrolase mRNA was ubiquitously expressed in the human cell lines, HeLa, SKG-IIIa, FL, KB, HEp-2, U373 GM, P3HR-1, Raji, THP-1, Jurkat, and Molt-4. These results suggested that BLM hydrolase plays important physiological roles, including the metabolism of antibiotics.

Bufalin, an active principle of Chinese medicine, chan'su, induced typical apoptosis in human leukemia U937 cells. When U937 cells were treated with 10(-8) M bufalin in the absence of serum, mitogen-activated protein (MAP) kinase activity was markedly increased 6 h after the start of treatment and elevated so for 12 h. Prior to the activation of MAP kinase, increased activities of Ras, Raf-1, and MAP kinase kinase were found, but these enzymes were transiently activated by the treatment with bufalin. These results suggest that the signal was transmitted sequentially from Ras, Raf-1, and MAP kinase kinase to MAP kinase. In association with this signal transduction, the concentration of cAMP in the cells decreased markedly, suggesting that Raf-1 was also activated by a decrease in the extent of phosphorylation by protein kinase A. In fact, pretreatment of U937 cells with forskolin and 3-isobutyl-1-methylxanthine, which are known to increase the concentration of cAMP in the cells, and subsequent treatment with bufalin resulted in a decrease in both Raf-1 activity and DNA fragmentation. To confirm the participation of MAP kinase in the apoptotic process, antisense cDNA for MAP kinase kinase 1 was expressed in U937 cells. The transformants were significantly resistant to both DNA fragmentation and cell death in response to bufalin. Our findings suggest that a pathway with the persistent activation of MAP kinase in U937 cells in response to bufalin is at least one of the signal transduction pathways involved in the induction of apoptosis.

Bleomycin (BLM) hydrolase, which hydrolyzes the carboxyamide bond in the beta-amino-alanine moiety, was purified from newborn rat skin. The enzyme was purified 2,500-fold over the crude extract to apparent homogeneity in five steps in the presence of 2-mercaptoethanol: 45-55% ammonium sulfate fractionation, followed by chromatographies on Sephacryl S-200, DEAE-cellulofine, Phe-Superose, and Mono Q ion-exchange. The native enzyme had a molecular mass of 280 kDa according to gel filtration. The subunit molecular mass was estimated as 48 kDa by SDS-PAGE, indicating that the enzyme was comprised of six identical subunits. The amino acid sequence of its NH2-terminus was determined to be acetyl-Met-Asn-Asn-Ala-Gly-Leu-Asn-Ser-Glu-Lys-, which was not found in the amino acid sequence database. The optimum pH of the enzyme was 7.5 with pepleomycin (PLM). The Km and Vmax values were 2.1 mM and 6.8 mu mol center dot mg-1 center dot h-1 for PLM, and 1.8 mM and 7.2 mu mol center dot mg-1 center dot h-1 for BLM-A2, respectively. The enzyme activity was inhibited by iodoacetic acid, N-ethylmaleinimide (NEM), and p-chloromercuribenzoic acid (pCMB) as well as divalent cations such as Cu2+, Cd2+, Hg2+, and Zn2+. It was effectively inhibited by a cysteine protease inhibitor E-64. However, cystatins A and C did not inhibit the activity. BLM hydrolase exhibited broad aminopeptidase substrate specificity towards aminoacyl-beta-naphthylamides such as basic, neutral, and hydrophobic amino acid residues, as well as acidic residues. These results indicated that BLM hydrolase represents a new family of cysteine proteases. Western blotting and immunohistochemical analyses showed that BLM hydrolase is ubiquitous in various rat tissues but at low levels in lung and adult skin tissues, suggesting that this enzyme plays an important role in the metabolism of antibiotics.

To study the function of acyl-CoA oxidase in an n-alkane-assimilating yeast, Candida maltosa, we isolated the POX2 gene which is a member of the acyl-CoA oxidase gene family. POX2 had a 2172-bp open reading frame (ORF) encoding an approx. 84-kDa polypeptide (724 amino acids (aa)) and was contiguous to POX4, another member of the acyl-CoA oxidase gene family on the same chromosomal DNA in a convergent arrangement. Northern blot analysis revealed that the expression of POX2 was induced in cells grown on oleic acid, n-tetradecanol and n-tetradecane. By using a gene-disruption technique, we constructed strains (termed P2DD and P4DD) in which both alleles of POX2 and POX4 were disrupted. The P2DD strain was normal in assimilation of various hydrophobic carbon sources, such as n-tetradecane, n-tetradecanol and oleic acid. In contrast, the P4DD strain was defective in its ability to grow on such hydrophobic carbon sources.

A low concentration of bufalin, a component of bufadienoides in the traditional Chinese medicine chan'su, was shown previously to induce differentiation of a broad range of human leukemia cell lines. In the present study, we found that bufalin at concentrations of 10(-7) M and higher induced apoptosis in human leukemia cells, such as HL60, ML1, but not in mouse leukemia M1 cells. A mere 15 min pretreatment of HL60 cells with 10(-6) M bufalin, followed by incubation for 15 h without bufalin, caused fragmentation of DNA and a decrease in cell viability, indicating that the signal for induction of apoptosis is triggered rapidly upon treatment with bufalin. Bufalin-induced apoptosis in HL60 cells was inhibited by ZnCl2, an inhibitor of endonuclease, but not by cycloheximide, an inhibitor of protein synthesis. Northern blot analysis revealed that the levels of expression of the c-myc and bcl-2 genes in HL60 cells decreased with time after treatment with bufalin. These results suggest that bufalin induces apoptosis specifically in human leukemia cells by altering the expression of these genes involved in apoptosis.

The aim of the study was to determine the optimal size and technique for construction of the systemic-to-pulmonary arterial shunt which will provide suitable pulmonary blood flow in first-stage Norwood palliation for hypoplastic left heart syndrome in neonates. Our clinical experience suggested that an arterial oxygen tension of about 30 mm Hg immediately after cardiopulmonary bypass, with the patients being ventilated at the lowest possible mean airway pressure with an FiO2of 1.0, provided a suitable pulmonary-to-systemic flow ratio. We also aimed to clarify the characteristics of pulmonary blood flow in accordance with the size of the shunt and the change in the pulmonary vascular resistance in a simplified rigid model of the Norwood procedure. A hole of2.0 mm diameter proved adequate to provide a suitable pulmonary blood flow of 200−300 mlx002F;min in the presence of a pressure gradient of 20−40 mm Hg between the systemic and pulmonary circulations in neonates weighing 3 kg. A short central shunt with a prosthesis of4 mm in diameter produced an excessive flow of pulmonary blood. Our data suggest that using a smaller shunt than that commonly used is necessary to decrease the early and intermediate postoperative mortality. A prosthesis of 3.0 or 3.5 mm in diameter arising from the brachiocephalic artery would be acceptable and can be recommended for first-stage Norwood palliation in small infants, especially in view of the operative difficulties encountered in taking down the shunt at the time of subsequent operations.

An alkane-assimilating yeast Candida maltosa had been studied in order to establish systems suitable for biotransformation of hydrophobic compounds. However, functional expression of heterologous genes tested for this purpose had not been successful in several cases. On the other hand, it had been reported that the codon CUG, a universal leucine codon, is read as serine in C. cylindracea. The same altered codon usage had also been suggested by in vitro experiments in some Candida yeasts which are phylogenetically closely related to C. maltosa. In this study we have shown that the failure in functional expression of a heterologous gene is due to the fact that the codon CUG is read as serine in C. maltosa. This conclusion was drawn from the following experimental results: (1) when a cytochrome P450 gene of C. maltosa containing a CTG codon was expressed in C. maltosa, the corresponding amino acid was found to be serine, and not leucine; (2) a tRNA gene with an almost identical structure to that of the tRNA SerCAG gene of C. albicans could be isolated from the genome of C. maltosa; (3) the Saccharomyces cerevisiae URA3 gene, which has one CTG codon, could not complement the ura3 mutation of C. maltosa as itself, but when the CTG codon was changed to another leucine codon, CTC, the mutated gene could complement the ura3 mutation. The last result is the first example of succeeding in functional expression of a heterologous gene in Candida species having an altered codon usage by changing the CTG codon in the gene to another codon. The nucleotide sequence datum reported in this paper will appear in the GSDB, DDBJ, EMBL and NCBI nucleotide sequence databases with the Accession Number D26074.

The effect of stimulation of the muscarinic receptor on Ca 2+ mobilization in cultured bovine adrenal chromaffin cells was examined. Acetylcholine (ACh) increased the uptake of 45 Ca 2+ and [Ca 2+ ] i whose levels decreased with time after reaching peaks. It also enhanced the efflux of 45 Ca 2+ from the cells. Its effect was inhibited by the specific muscarinic receptor antagonist atropine (Atr), but not by the nicotinic receptor antagonist hexamethonium (C 6 ). The increase in muscarine (Mus)-stimulated 45 Ca 2+ efflux was reduced concentration-dependently by deprivation of extracellular Na + . These results suggest that muscarinic stimulation of the ACh receptor stimulates Na + /Ca + exchange in cultured bovine adrenal chromaffin cells.

We found that bufalin, an active principle of the Chinese medicine chan'su, has selective inhibitory effects on the growth of various human cancer cells. In order to examine whether the growth-inhibitory effect of bufalin on human cancer cells is associated with apoptosis, human leukemia cells were treated with bufalin. HL-60, ML1, and U937 leukemia cells treated with bufalin at 10(-8) M and above had condensed and fragmented nuclei. Flow cytometric analysis of these cells treated with bufalin showed fragmented DNA smaller than that of the G1 phase. DNA of HL-60 cells treated with bufalin showed a ladder pattern characteristic of apoptosis, as analyzed by agarose gel electrophoretic analysis. DNA synthesis and topoisomerase II activity of HL-60 cells were markedly inhibited as the concentration of bufalin was increased. The concentration needed for inducing apoptosis of HL-60 cells was 10(-8) M, which is comparable to that of camptothecin, but lower than those of other antitumor drugs such as cisplatin, VP16 and all-trans retinoic acid. Apoptosis was not observed when human mononuclear and polymorphonuclear cells were treated with 10(-6) M bufalin for 24 h. These results indicate the association of the growth-inhibitory effect of bufalin with the induction of apoptosis, at least in HL-60 cells, and suggest the usefulness of bufalin for differentiation-apoptosis-inducing therapy for cancer.

A gene encoding phosphoglycerate kinase (PGK) was isolated from the genomic library of C. maltosa to construct an expression vector for this yeast. The PGK gene had an open reading frame of 1,251 base pairs encoding approximately 47-kDa polypeptide of 417 amino-acid residues. Expression of this gene assayed by Northern-blot analysis was significantly induced in cells grown on glucose but not in cells grown on n-tetradecane, n-tetradecanol, or oleic acid. By using the promoter region of this gene, an expression vector (termed pMEA1) for C. maltosa was constructed and expression of an endogenous gene (P450alk1 encoding one of cytochrome P450s for n-alkane hydroxylation in C. maltosa) and a heterologous gene (LAC4 encoding Kluyveromyces lactis beta-galactosidase) was tested. Expression of P450alk1 gene was confirmed at both mRNA and protein levels. LAC4 gene expression was confirmed by determining beta-galactosidase activity. The activity in cells grown on various carbon sources correlated very well with the expression levels of PGK mRNA in these cells.

The effects of two K+ channel openers, cromakalim and pinacidil, on voltage-dependent and receptor-mediated catecholamine secretion and Ca2+ mobilization in bovine adrenal chromaffin cells were studied to determine the role of membrane K+ channels in the regulation of a Ca(2+)-dependent secretory process. Both cromakalim and pinacidil stimulated the efflux of 86Rb (used to monitor K+ permeability) from preloaded cells. Cromakalim and pinacidil did not affect the catecholamine secretion induced by excessive depolarization with 56 mM K+, but inhibited that induced by moderate depolarization with 31 mM K+ in a concentration-dependent manner (1 microM-100 microM). The 31 mM K(+)-induced 45Ca2+ influx and increase in intracellular free Ca2+ concentration [Ca2+]i were also inhibited by these agents at similar concentrations to those for inhibition of catecholamine secretion. Cromakalim and pinacidil inhibited catecholamine secretion, 45Ca2+ influx and increase in [Ca2+]i induced by stimulation of nicotinic acetylcholine (ACh) receptors with carbamylcholine. Furthermore, both cromakalim and pinacidil inhibited the increase in [Ca2+]i induced by carbamylcholine in the absence of extracellular Ca2+, which is thought to be mediated by muscarinic ACh receptors. On the other hand, they did not affect catecholamine secretion induced by Bay-K 8644, Ba2+, A23187, histamine or bradykinin. These results indicate that the K+ channel openers, cromakalim and pinacidil, selectively inhibit catecholamine secretion induced by moderate depolarization or by nicotinic ACh receptor stimulation by inhibiting Ca2+ influx and increase in [Ca2+]i. Furthermore, the results suggest that these K+ channel openers-sensitive membrane K+ channels are involved in the regulation of catecholamine secretion mainly indirectly through effects on the voltage-dependent membrane Ca2+ channels.

Background and Objectives We describe the use of mandibular nerve block for the management of bilateral trismus associated with hypoxic-ischemic encephalopathy. Case Report The patient was a 65-year-old man with bilateral trismus due to hypoxic-ischemic encephalopathy. Despite his impaired consciousness, we performed fluoroscopically guided bilateral mandibular nerve block. The bilateral symptoms were sufficiently improved, without obvious side effects, by injecting a local anesthetic near the right mandibular nerve and a neurolytic near the left mandibular nerve. Conclusions Mandibular nerve block may be an effective treatment for patients with bilateral trismus due to ischemic-encephalopathy, even when consciousness is impaired.

We detected a peptide having a behavioral activity on mouse forced swimming from sera of healthy volunteers without affective and psychotic diseases. The amino acid sequence was GVNDNEEGF, which was found in the sequence of human β-fibrinogen. The synthesized peptide also showed the behavioral activity dose-dependently, but human fibrinopeptide B (QGVDNEEGFFSAR) did not. The activity was decreased by dopamine 1 antagonist SCH-23390, but not by dopamine 2 antagonist sulpiride. These findings strongly suggest that metabolism of human β-fibrinogen induces the fragment affecting the mouse behavior via the dopamine1 neuronal activity.

The treatment of depression in senile patients with dementia is difficult with the drugs used formerly. The effects of a new anxiolytic drug, tandospirone, were investigated on depression symptoms in nine senile patients with dementia using Hamilton Depression Rating Scale (HAM-D) items. Tandospirone improved the symptoms, especially the depressive mood, agitation and anxiety, although a slight gastrointestinal symptom was found in one patient. The findings in the present study may suggest that tandospirone is a useful and comparatively safe drug for depression symptoms in senile patients with dementia.

To evaluate the usefulness of plasma dopamine-beta-hydroxylase (DBH) activity as an index of sympathetic nerve activity during cardiac operations, we examined the serial changes in plasma DBH activity, in relation to the plasma noradrenaline (NA) level and hemodynamic parameters, in patients who underwent cardiac surgery. The plasma DBH activity decreased significantly after cardiopulmonary bypass, and remained low during dopamine (DA) infusion until 72 h after the operation. However, recovery of the hemodynamic parameters, being the mean arterial pressure, heart rate and cardiac index, was seen as early as 1-3 h postoperatively. It was therefore assumed that the plasma DBH activity takes a long time to recover after an operation. The time-course changes in the plasma NA level were quite different from the changes in DBH activity, with an apparent negative correlation being observed between them. Thus, there is a possibility that exogenously administered DA, as well as increased plasma NA, might inhibit DBH activity during cardiac surgery. Moreover, since catecholamines are often administered upon completion of cardiac surgery, measurement of the plasma catecholamine level would be inappropriate for evaluating real sympathetic nerve activity. From the results of this study, it is surmised that measurement of the plasma DBH activity could be useful for estimating the intrinsic sympathetic nerve activity of patients who have undergone cardiac surgery.

We examined the efficacy and safety of a new transdermal fentanyl citrate patch (HFT-290), which was applied once daily in patients with cancer pain who were receiving a stable dose of once-every-three-day application transdermal fentanyl patch [TDF (72 hr)]. After TDF (72 hr) was applied for three days at the same dose used before starting the study, treatment was switched to HFT-290 (once daily) for 9 days. The analgesic effect was judged with a 5-point scale based on each patient's assessment of pain on a 100-mm visual analog scale (VAS). Seventy-eight patients were enrolled. The efficacy rate (95% confidence interval) of the analgesic effect at the time of final removal of HFT-290 (the primary efficacy end-point) was high at 83.9% (71.7-92.4%; 47/56 patients). Furthermore, based on the shift of the VAS, good pain control was achieved after switching. All adverse drug reactions were either mild or moderate, and the main reactions were those commonly observed with opioid analgesics. No respiratory depression was observed. HFT-290 demonstrated good tolerability after switching from TDF (72 hr) and provided stable pain control.

In many cases, a new railway line is planned beneath existing structures of an urban terminal station. The underpinning method has been developed to respond to such needs. However, this conventional method involves a number of steps and is costly and time-consuming. The steps are: (1) Constructing the cast- in-situ diaphragm walls to keep off the underground water; (2) Supporting the existing structures with the cast- in-situ piles; (3) Excavating the soil under the existing structures; (4) Constructing structures for the new railway; (5) Transferring the loads of the existing structures from the temporary piles onto the newly constructed structure; and (6) Removing the temporary piles. In introducing a new subway line beneath Nagoya station, which is one of the major terminals in Japan, the Central Japan Railway Company made use of temporary structures, cast- in-situ diaphragm walls and piles, as elements of the new structures adopting new measures to increase the accuracy of underground construction. It is suggested that this method can save considerable construction time and cost.

イオンクロマトグラフィーによる市販ミネラルウォーター製品44試料,及び,そのほかの飲料水10試料中の8主要無機イオン(Na+, K+, Mg2+, Ca2+, Cl-, NO3-,SO42-, HCO3-)分析,及び,モリブデン青法による溶性ケイ酸分析を行った.また,水のおいしさについて官能検査を行った.(1) 無機成分の組成及び含有量は試料により異なっていた.無機成分総量の平均値は,国産ミネラルウォーター製品,輸入ノンガスタイプミネラルウォーター製品,輸入ガスタイプミネラルウォーター製品,水道水,井戸水が,各々, 153, 322, 1856, 189, 175ppmであった.輸入ガスタイプミネラルウォーター製品はHCO3-含有量が多かった.国産ミネラルウォーター製品と水道水及び井戸水の無機成分組成に大きな違いは無かった.(2) ミネラルウォーター製品及び水道水の官能検査の結果,水のおいしさに影響を与える要因は,本報で分析した試料でみられた程度の無機成分組成の違いではなく,むしろ,かび臭やカルキ臭であった.

A specific dopamine 2 receptor antagonist, (−)sulpiride, induced an anti-depressive behavior, climbing, in mice forced to swim for 6 h after the injection. The effective fraction was divided from the mouse serum using an ion exchanger and an ultra filtration method. This fraction contained fibrinopeptide A. A peptide synthesized according to the primary 6-amino acid sequence (TDTEDK) of fibrinopeptide A also remarkably increased the behavior. The present findings clearly indicate that a peptide with TDTEDK showed anti-depressive activity.

SummaryThrombin-activatable fibrinolysis inhibitor (TAFI), a carboxypeptidase B-like proenzyme, is predominantly biosynthesised in the liver and released into circulating plasma. Activated TAFI has a role in maintaining the balance between blood coagulation and fibrinolysis. We investigated the regulation of TAFI expression in cultured human hepatoma HepG2 cells. Stimulation of the cells with forskolin and dibutyryl cyclic AMP (DBcAMP) increased TAFI antigen levels in the cells in parallel with TAFI mRNA levels and antigen release from the cells into the conditioned medium. The elevated TAFI expression was abolished by pretreatment of the cells with KT5720, a protein kinase A (PKA) inhibitor. The promoter activity of the TAFI gene and the half-life of the TAFI transcript in DBcAMP-stimulated HepG2 cells increased to 1.5-fold and 2.0-fold, respectively, of those in the control cells. The increased promoter activity and the prolonged half-life were abolished by pretreatment of the cells with KT5720.These results suggest that an increase in intracellular cAMP levels upregulates TAFI expression in the cells in accompaniment with an elevation of TAFI mRNA levels, and that the elevated mRNA levels are derived from both transcriptional and post-transcriptional regulations of the TAFI gene mediated by activation of the AMP/PKA signaling pathway.

The objective of this study was to investigate relationships between reproductive traits in heifers and cows and yield traits for Holsteins in Japan. Insemination and lactation records for cows calved between 1990 and 2003 in Hokkaido region were obtained. Age at first service, age at conception, and conception rate for first service were calculated for heifers. Days from calving to first service, days open, and conception rate for first service were calculated for first- and second-parity cows. The yield traits used were 305-d milk, fat, and protein yields. A threshold animal model was applied for the conception rate for first service, and a linear animal model was applied for the other traits. Single-trait and 2-trait genetic analyses were performed by the Bayesian method using Gibbs sampling. Heritability estimates ranged from 0.027 to 0.051 for conception rate for first service, and from 0.074 to 0.128 for the other reproductive traits. If the relationships of other traits were not considered, days from calving to first service was favorable to genetic selection for reproductive traits because of relatively high heritability and because it can be available earlier than the days open. Genetic correlations among reproductive traits were high, especially in cows. The genetic correlations between reproductive traits for heifers and those for cows were lower than the genetic correlations between reproductive traits for first parity and those of second parity, suggesting that reproductive traits for heifers should be evaluated separately from reproductive traits for cows. Genetic correlations between yield and reproductive traits in cows were antagonistic. In contrast, genetic correlations between reproductive traits for heifers and yield traits were slightly desirable. Depending on the reporting rate of insemination records for heifers and the results of investigations for relationships with productive maturity, selection by reproductive traits for heifers will enable the improvement of reproductive performance without a loss in genetic progress for yield traits.

Adipose differentiation-related protein (ADRP) is a major protein localized at the lipid droplets in macrophage-derived foam cells or liver cells. However, the role of ADRP during regression of lipid-storing cells has not been understood. When J774 macrophages were incubated for 3 days with VLDL, their content of ADRP and triacylglycerol (TG) increased 3- and 4-fold, respectively. Induction of ADRP was observed by loading lipids in macrophages with either oleic acid, suggesting that the induction ADRP induction was not receptor-dependent. ADRP expression during TG accumulation was also induced in oleic acid-treated HuH-7 human liver cells. As TG decreased in the foam cells by treatment with triacsin C, an acyl-CoA synthase inhibitor, for 6 h, ADRP protein decreased in parallel. Treatment of lipid-stored HuH-7 cells with triacsin C also reduced ADRP, indicating that ADRP reduced during regression of the lipid-storing cells. Such decrease in ADRP during regression of TG-storing cells was abolished by co-incubation with a proteasome inhibitor. Poly-ubiquitinated ADRP was detected by a pull-down experiment in the presence of the proteasome inhibitor. In addition, the proteasome inhibitor reversed not only the degradation of ADRP but TG loss by triacsin C in HuH-7 cells. The amount of ADRP is reciprocally regulated with the lipid content in the cells, and the ubiquitin-proteasome system is involved in degradation of ADRP during regression of lipid-storing cells.

To elucidate the mechanism of palytoxin (PTX)-induced Na+ influx, we examined the effect of amiloride, an inhibitor of Na+/H(+)-antiporter, on PTX-induced Na+ influx into cultured bovine adrenal chromaffin cells in relation to its effects on Ca2+ influx and catecholamine secretion. Amiloride dose-dependently inhibited PTX-induced 22Na+ influx, whereas tetrodotoxin (TTX) had no effect. Amiloride also inhibited PTX-induced Na(+)-dependent 45Ca2+ influx and catecholamine secretion. PTX alone did not significantly affect the intracellular pH, but it decreased in the presence of PTX and amiloride. These results indicate that an amiloride-sensitive Na+/H+ exchange mechanism is probably involved in PTX-induced, TTX-insensitive Na+ influx that triggers Ca2+ influx and catecholamine secretion from the cells.

市販バニラアイスクリーム及び原材料の乳製品と卵黄について,全自動高速アミノ酸分析機により遊離アミノ酸を定量した.(1) バニラアイスクリーム11試料の遊離アミノ酸総量は94～338μg/gで,主要な遊離アミノ酸はグルタミン酸,タウリン,フォスフォエタノールアミンであった.(2) アイスクリームを価格帯により4グループに分類し,価格の高い順にスーパープレミアムタイプアイスクリーム,プレミアムタイプアイスクリーム,通常のアイスクリーム,ラクトアイスとした.価格帯の高いグループほど遊離アミノ酸総量が多かった.遊離アミノ酸のパターンは,スーパープレミアムタイプアイスクリームのみ他のグループと異なっていた.これは,卵黄由来の遊離アミノ酸によると推定された.(3) 原材料の遊離アミノ酸総量は,生クリーム,バタ一,脱脂濃縮乳,脱脂粉乳,卵黄が,各々,65, 42, 470, 1196, 4697μg/gであった.バニラアイスクリームに使用されている乳製品は製造工程中の加熱履歴が異なるが,無脂乳固形分当たりの遊離アミノ酸総量も組成もほとんど変わらなかった.スーパープレミアムタイプアイスクリーム以外のグループの遊離アミノ酸組成は,乳製品と類似していた.

Surface dose monitoring in patients and physicians during 29 uterine artery embolisation (UAE) procedures was performed using photoluminescence dosemeters and thermo-luminescence dosemeters. Organ or tissue doses were measured with an anthropomorphic phantom using UAE exposure conditions averaged from the 29 cases, and effective doses were estimated for the patient. Entrance surface dose of the patients at the maximum dose position ranged from 121.5 to 1650 mGy. Estimated doses ranged from 3.16 to 43 mGy for the ovary and from 3.8 to 51.8 mGy for the uterus. The effective dose was 1.09-14.8 mSv. Monitored doses on the body surface of physicians were relatively high in the upper arm (5.41+/-1.52 to 163+/-17.25 microGy) and the hand and fingers (0.85+/-1.18 to 222+/-16.4 microGy).

This paper describes the morphometric analysis of preoperative aspiration materials from breast cancers to define cytological predictors of recurrence and malignancy grade. Subjects were 65 patients with primary invasive ductal carcinoma of the breast, of which 36 were cancer free at least for 5 years from radical operation (cancer free group) and 29 had distant recurrences in 5 years (recurrence group). Morphological factor of nuclei was calculated for each group. Nuclear area itself had no relation with prognosis. Coefficiency variant of nuclear area (NA·CV) was calculated. NA·CV of recurrence group was higher than that of cancer free group, as shown in these figures, 39.8±7.9% and 25.5±4.6% respectively. NA·CV was most variable prognostic indicator among the several prognostic factors, for example tumor size (T'), histological type, and lymph nodal status. By using morphometry and calculating NA·CV, cytological nuclear pleomorphism was evaluated objectively. The prognosis and malignancy grade of the breast cancer can be probably presumed at preoperative period.

Nobiletin is a citrus polymethoxylated flavonoid extracted from Citrus depressa, and has several reported biological effects. In this study, we investigated the effect of nobiletin on bacterial lipopolysaccharide (LPS)-induced expression of tissue factor (TF), a trigger protein for the blood coagulation cascade, and studied the possible mechanism of TF transcriptional regulation. THP-1 monocytic cells stimulated with LPS showed an increased expression of both TF protein and mRNA levels. However, pretreatment with nobiletin resulted in inhibition of LPS-induced expression of both TF protein and mRNA in a dose-dependent manner. Electrophoretic mobility shift assays revealed that binding of nuclear proteins from LPS-stimulated THP-1 cells to the NF-kappaB or AP-1 binding motif was increased as compared to non-stimulated control cells. Such increased binding activities were significantly reduced by pretreatment with nobiletin. Binding activity of nuclear proteins to the Sp1 binding motif was observed irrespective of LPS stimulation, but Sp1 activation was inhibited by nobiletin treatment of the cells. Treatment of THP-1 cells with Sp1-specific small interfering RNA (Sp1 siRNA) abolished the ability of LPS to induce TF activity. A similar reduction in the level of TF mRNA was also observed upon treatment of cells with Sp1 siRNA. These studies reveal that constitutive Sp1 activation is an essential event for transcriptional activation of TF, and nobiletin prevents LPS-induced TF expression by inhibiting NF-kappaB, AP-1, and Sp1 activation.

Apoptotic cell death was induced in human lung cancer DMS114 cells by treatment with beta-hydroxyisovalerylshikonin (beta-HIVS), an ATP-noncompetitive inhibitor of protein tyrosine kinases. Changes in phosphoprotein profiles were analyzed by two-dimensional-polyacrylamide gel electrophoresis (2D-PAGE) after the cells were treated with beta-HIVS. One spot on the 2D gel showed a marked decrease in intensity and the corresponding protein was identified by mass spectrometry as dUTP nucleotidohydrolase (dUTPase). The beta-HIVS-induced decrease of dUTPase in the phosphoprotein fraction of DMS114 cells was confirmed using immunoblotting. Treatment of the cells with beta-HIVS-induced rapid reduction of dUTPase activity. An antioxidant N-acetyl-cysteine inhibited both the reduction of phosphorylated dUTPase and the induction of apoptosis by beta-HIVS treatment of DMS114 cells. Introduction of siRNA directed against dUTPase mRNA into DMS114 cells enhanced the susceptibility of beta-HIVS-induced apoptosis. Treatment of DMS114 cells with beta-HIVS and 5-fluorouracil, a specific inhibitor of thymidylate synthase used as a chemotherapeutic drug, revealed the synergistic effects of these drugs on the inhibition of cell growth. These results suggest that dUTPase activity is one of the crucial factors involved in apoptotic cell death in lung cancer cells.

Oxidatively modified low-density lipoprotein (oxLDL) is one of the major factors involved in the development of atherosclerosis. Because of the insolubility of apolipoprotein B-100 (apoB-100) and the heterogeneous nature of oxidative modification, modified structures of apoB-100 in oxLDL are poorly understood. We applied an on-Membrane sample preparation procedure for LC-MS/MS analysis of apoB-100 proteins in native and modified low-density lipoprotein (LDL) samples to eliminate lipid components in the LDLs followed by collection of tryptic digests of apoB-100. Compared with a commonly used in-gel digestion protocol, the sample preparation procedure using PVDF membrane greatly increased the recovery of tryptic peptides and resulted in improved sequence coverage in the final analysis, which lead to the identification of modified amino acid residues in copper-induced oxLDL. A histidine residue modified by 4-hydroxynonenal, a major lipid peroxidation product, as well as oxidized histidine and tryptophan residues were detected. LC-MS/MS in combination with the on-Membrane sample preparation procedure is a useful method to analyze highly hydrophobic proteins such as apoB-100.

It has been previously demonstrated that bufalin, an active agent in the Chinese medicine chan'su, induces apoptosis in human leukemia cells by altering the expression of apoptosis-related genes, such as bcl-2 and c-myc. Tiam1 was also found to play a critical role in bufalin-induced apoptosis through the activation of the Rac1, PAK and JNK pathway in human leukemia cell lines. In the present study, the involvement of the Tiam1 gene products in bufalin-induced apoptosis in human solid tumor HeLa cells was examined.HeLa cells were treated with 10(-8) M bufalin and apoptosis was measured by ELISA quantification of nucleosomes. Tiam1 mRNA levels were quantified by real-time PCR analysis and inhibited by transfected siRNA specific for Tiam1.Apoptosis was induced in HeLa cells by treatment with 10(-8) M bufalin. Expression of both Tiam1 mRNA and its protein was induced 0.5 h after the start of the bufalin treatment. Transfection of Tiam1-specific siRNA into HeLa cells markedly inhibited bufalin-induced apoptosis.Our results suggest that Tiam1 is a downstream mediator of bufalin-induced apoptosis in the human solid tumor HeLa cell line, as well as in leukemia cell lines.

Sleep bruxism (SB) has been believed to be related to stress and psychosocial factors, however their implicit relationship has remained unclear. This study was conducted on patients visiting our clinic with SB and masticatory muscle disorders (MMD) for the purpose of clarifying personality and behavioral traits. This study was conducted on patients of MMD visiting our clinic. The Rosenzweig Picture-Frustration study was performed on each patient. Twenty-seven (27) patients were divided into two groups: 17 patients with SB and 10 patients without. The SB group showed a significantly lower level of E (extrapunitive) reaction than the nonSB group. SB patients showed a significantly higher level of M (impunitive) reaction than those without SB. Concerning the directions of aggression, the percentage of E-A (extraaggression) was significantly lower in SB patients than in those without. On the other hand, the percentage of I-A (intraaggression) was significantly higher in patients with SB than those without. Our study found a new aspect of the patients with MMD and SB: they are not only intraaggressive, but are also unable to be extrapunitive and extraaggressive. Consequently, they are unable to demonstrate adequate self-assertiveness in stressful situations.

Geranylgeraniol (GGO) induces apoptosis in various lines of human tumor cells through a mitochondrion-dependent pathway. The present study describes identification of a 21-kDa cytochrome c-releasing factor that appears in the cytosolic fraction after treatment of human leukemia U937 cells with GGO. Incubation of isolated mitochondria with a lysate of U937 cells that had been treated with GGO resulted in the release of cytochrome c from the mitochondria. Utilizing this cell-free system, we purified a 21-kDa protein that induced the release of cytochrome c from mitochondria and appeared to be involved in the apoptosis that is induced in U937 cells by GGO. We designated this protein cytochrome c-releasing factor 21 (CRF21). Overexpression of CRF21 in HeLa cells induced the release of cytochrome c from mitochondria, with subsequent apoptosis. Our results suggest that CRF21 might play an important role in the induction of apoptosis by GGO in leukemia U937 cells.

Adipose differentiation-related protein (ADRP) is a major protein associated with lipid droplets in various types of cells, including macrophage-derived foam cells and liver cells. However, the role of ADRP in the processes of formation and regression of these cells is not understood. When J774 murine macrophages were incubated with either VLDL or oleic acid, their content of both ADRP and triacylglycerol (TG) increased 3- to 4-fold. Induction of ADRP during TG accumulation was also observed in oleic acid-treated HuH-7 human liver cells. Addition of triacsin C, a potent inhibitor of acyl-CoA synthase, for 6 h decreased the amount of TG in VLDL-induced foam cells and oleic acid-treated liver cells; it decreased the amount of ADRP protein in parallel, indicating the amount of ADRP reduced during regression of the lipid-storing cells. Addition of a proteasome inhibitor during triacsin C treatment abolished the ADRP decrease and accumulated polyubiquitinated ADRP. In addition, the proteasome inhibitor reversed not only the degradation of ADRP but also TG reduction by triacsin C. These results suggest that cellular amounts of ADRP and TG regulate each other and that the ubiquitin-proteasome system is involved in degradation of ADRP during regression of lipid-storing cells.

A cDNA clone encoding a heat-stable sweet protein, mabinlin II (MAB), was isolated and sequenced. The encoded precursor to MAB was composed of 155 amino acid (aa) residues, including a signal sequence of 20 aa, an N-terminal extension peptide of 15 aa, a linker peptide of 14 aa and one residue of C-terminal extension. Comparison of the proteolytic cleavage sites during post-translational processing of MAB precursor with those of like 2S seed-storage proteins of Arabidopsis thaliana, Brassica napus and Bertholletia excelsa shows that the three individual cleavage sites between respective species are conserved.

It is reported that high-dose methamphetamine (8 mg/kg) induces stereotyped behavior in ddY mice in an open field, but it is still not certain that low-dose methamphetamine (less than 2 mg/kg) can induce the stereotyped behavior in ddY mice in a narrow space. In order to investigate the problem, we evaluated the quantity of stereotyped behavior of ddY mice by using a mouse wheel-running apparatus. In this method, we have come to recognize an increase in the stereotyped behavior depending on the dose of methamphetamine and the reverse-tolerance phenomenon as a decrease in the wheel-revolution counts. The present findings indicate that low-dose methamphetamine can promote stereotyped behavior in ddY mice under conditions in which the ambulation is restricted to a narrow wheel space.

Curculin from Curculigo latifolia is a unique sweet protein that exhibits both sweet-tasting and taste-modifying activities. We isolated a gene that encodes a novel protein highly homologous to curculin. Using cDNAs of the previously known curculin (designated as curculin1) and the novel curculin isoform (curculin2), we produced a panel of homodimeric and heterodimeric recombinant curculins by Escherichia coli expression systems. It was revealed that sweet-tasting and taste-modifying activities were exhibited solely by the heterodimer of curculin1 and curculin2.