Quality control of hospital preparations: Establishment of a simple and rapid method for quantifying ulinastatin in vaginal suppositories.

Ulinastatin vaginal suppositories, used to prevent threatened premature delivery, are frequently used in hospitals. However, there is no established method for quantifying ulinastatin contained in suppositories. Therefore, we investigated a simple and efficient method for quantifying ulinastatin contained in suppositories. Our analytical method involved removal of the base; optimising the enzyme inhibition reaction time and enzyme reaction time; and measuring the absorbance. The modified method was reproducible, operation time was significantly shortened, and cost was reduced to approximately 1/17 of that of the previously reported method. This simple and rapid quantitative method could contribute to the improvement of quality control of ulinastatin vaginal suppositories as an extemporaneous hospital preparation.

Biomarkers for the evaluation of immunological properties during the shikoku walking pilgrimage.

It is important to determine the immunological properties for the maintenance of health. We chose the Shikoku Walking Pilgrimage to assess the proper biomarkers for the evaluation of immunological properties. We examined whether the Shikoku Walking Pilgrimage could have a positive effect on the mental and physical health of walking participants by using several biomarkers proposed by our laboratory. Twelve non-randomized healthy male volunteers including 3 twice attendees walked the Shikoku Walking Pilgrimage distance of 58.9 km over 3 days. Plasma, serum, urine, and saliva were collected from the volunteers during the pilgrimage and at 1 week before and after it. Immunological biomarkers, including lipid metabolism, oxidative stress, immune function, and catecholamines, were measured. Additionally, mood state scores, alertness, autonomic nervous system activity, and body motion levels during sleep were assessed. A significant decrease was observed in the subjective tension-anxiety levels and in the concentrations of serum low-density lipoprotein cholesterol, plasma hydroxyoctadecadienoic acid (HODE), and urine adrenaline during the pilgrimage as compared to the values of these parameters before the participants embarked on the pilgrimage. The serum levels of brain-derived neurotrophic factor (BDNF) were significantly increased 1 week after the pilgrimage relative to those assessed previously. No significant differences in subjective fatigue and the flicker perception threshold were observed. These results suggest that the Shikoku Walking Pilgrimage can exert a positive effect on mental and physical health as particularly shown in the reduction of tensionanxiety and oxidative stress without the accompaniment of fatigue. HODE correlated significantly with typical immunological marker natural killer cell activity and immunoglobulin G. This suggests that there are promising biomarkers such as HODE, NK activity, BDNF, LDL-c, and IgG for assessing the immunological properties.

A cohort study of racing performance in Japanese Thoroughbred racehorses using genome information on ECA18.

Using 1710 Thoroughbred racehorses in Japan, a cohort study was performed to evaluate the influence of genotypes at four single nucleotide polymorphisms (SNPs) on equine chromosome 18 (ECA18), which were associated in a previous genome-wide association study for racing performance with lifetime earnings and performance rank. In males, both g.65809482T>C and g.65868604G>T were related to performance rank (P= 0.005). In females, g.65809482T>C (P = 1.76E-6), g.65868604G>T (P=6.81E-6) and g.66493737C>T (P=4.42E-5) were strongly related to performance rank and also to lifetime earnings (P < 0.05). When win-race distance (WRD) among all winning racehorses and best race distance (BRD) among elite racehorses were considered as the phenotypes, significant associations (P<0.001) were observed for all four SNPs. The favourable race distance of both elite (BRD) and novice racehorses (WRD) was also associated with genotypes in the ECA18 region, indicating the presence of a gene in this region influencing optimum race distance in Thoroughbred racehorses. Therefore, the association with performance rank is likely due to the bias in the race distances. The location of the SNPs within and proximal to the gene encoding myostatin (MSTN) strongly suggests that regulation of the MSTN gene affects racing performance. In particular, the g.65809482T>C, g.65868604G>T and g.66493737C>T SNPs, or their combinations, may be genetic diagnostic markers for racing performance indicators such as WRD and BRD.

Accelerated neutrophil apoptosis in mice lacking A1-a, a subtype of the bcl-2-related A1 gene.

To elucidate the role of A1, a new member of the Bcl-2 family of apoptosis regulators active in hematopoietic cell apoptosis, we established mice lacking A1-a, a subtype of the A1 gene in mice (A1-a-/- mice). Spontaneous apoptosis of peripheral blood neutrophils of A1-a-/- mice was enhanced compared with that of either wild-type mice or heterozygous mutants (A1-a+/- mice). Neutrophil apoptosis inhibition induced by lipopolysaccharide treatment in vitro or transendothelial migration in vivo observed in wild-type mice was abolished in both A1-a-/- and A1-a+/- animals. On the other hand, the extent of tumor necrosis factor alpha-induced acceleration of neutrophil apoptosis did not differ among A1-a-/-, A1-a+/-, and wild-type mice. The descending order of A1 mRNA expression was wild-type, A1-a+/-, and A1-a-/-. Taken together, these results suggest that A1 is involved in inhibition of certain types of neutrophil apoptosis.

Rat lymphoid cell lines producing human T cell leukemia virus. II. Constitutive expression of rat interleukin 2 receptor.

Three rat lymphoid cell lines (TARS-1, TARL-2, and TART-1) (12) transformed by human T cell leukemia/lymphoma virus I (HTLV-I) had rearrangement of the beta chain gene of the T cell antigen receptor, and had integrated proviral DNA from HTLV-I in their genomes. As is the case with adult T cell leukemia (ATL)-derived human T cell lines transformed by HTLV-I, these rat cell lines unequivocally expressed interleukin 2 (IL-2) receptor, as determined by radiolabeled IL-2 binding. By Scatchard plot analysis, one of the cell lines, TART-1, proved to have high affinity receptors (Ka = 1.3 X 10(11)/M and 8.8 X 10(9)/M). Rat IL-2 receptor, not human IL-2 receptor, was expressed on HTLV+ rat cell lines, as demonstrated by the fact that they expressed antigens reactive with monoclonal antibodies (ART-18) against rat IL-2 receptor, but not with anti-Tac antibodies. The collective evidence indicates that the endogenous IL-2 receptor gene is activated in human and rat lymphoid cell lines with HTLV-I production. The mechanism of abnormal IL-2 receptor expression in HTLV infection is discussed.

Suppressive effects of 1,4-dihydroxy-2-naphthoic acid administration on bone resorption.

SUMMARY: The main component of the metabolic by-products of fermentation by Propionibacterium freudenreichii ET-3 is 1,4-dihydroxy-2-naphthoic acid (DHNA), which has a naphthoquinone skeleton, as in vitamin K2. This study showed that DHNA improved bone mass reduction with osteoporosis model mice caused by FK506. INTRODUCTION: Growth of the intestinal bacterium Lactobacillus bifidus is specifically facilitated by DHNA. The present study used osteoporosis model mice to investigate the effects of DHNA on bone remodeling. METHODS: FK506, an immunosuppressant, was used to prepare osteoporosis model mice. Thirty mice were divided into three groups: FK group, FK+DHNA group, and control group. In the FK group, FK506 was administered to induce bone mass reduction. In the FK-DHNA group, FK506 and DHNA were administered concurrently to observe improvements in bone mass reduction. To ascertain systemic and local effects of DHNA, we investigated systemic pathological changes in colon, kidney function and cytokine dynamics, and morphological and organic changes in bone and osteoclast dynamics as assessed by culture experiments. RESULTS: Compared to the FK group without DHNA, colon damage and kidney dysfunction were milder for FK+DHNA group, and production of inflammatory cytokines (interleukin (IL)-1beta, IL-6 and tumor necrosis factor (TNF)-alpha) was more suppressed. Furthermore, compared to the group without DHNA, histological analyses and radiography showed that bone resorption was suppressed for the DHNA group. Culture experiments using osteoclasts from murine bone marrow showed osteoclast suppression for the DHNA group compared to the group without DHNA. CONCLUSION: These results show that DHNA has some effects for improving bone mass reduction caused by FK506.

Herpes zoster of the nipple: rapid DNA-based diagnosis by the loop-mediated isothermal amplification method.

A 28-year-old Japanese man presented with grouped erosions and vesicles on an erythematous base affecting the right areola and the surrounding skin. A Tzanck smear from the vesicle revealed giant cells. An initial clinical diagnosis of mammary herpes simplex was considered but to explore the differential diagnosis, viral DNA was amplified by the loop-mediated isothermal amplification (LAMP) method. DNA replication was observed only in varicella zoster virus LAMP mixture, and this confirmed a diagnosis of herpes zoster. The patient was treated with 3000 mg of daily oral valacyclovir for seven days. After antiviral treatment, the lesion had healed and the pain had resolved completely.

Disseminated mucocutaneous herpes simplex virus infection in an immunocompetent woman.

Disseminated mucocutaneous herpes simplex virus (HSV) infection in an immunocompetent person is quite rare. A 19-year-old healthy Japanese woman presented with painful, umbilicated vesicles and pustules on her genital region, both nipples and on the forearm 10 days after the last sexual contact with her partner who had cold sore at that time. Tzanck test and biopsy confirmed the diagnosis of disseminated mucocutaneous HSV infection. She did not have any visceral HSV disease. Skin lesions improved after treatment with acyclovir and erythromycin for seven days. We propose that like herpes gladiatorum, HSV dissemination in this case was acquired by close body contact.

A genome-wide scan for tying-up syndrome in Japanese Thoroughbreds.

Tying-up syndrome, also known as recurrent exertional rhabdomyolysis in Thoroughbreds, is a common muscle disorder for racehorses. In this study, we performed a multipoint linkage analysis using LOKI based on the Bayesian Markov chain Monte Carlo method using 5 half-sib families (51 affected and 277 nonaffected horses in total), and a genome-wide association study (GWAS) using microsatellites (144 affected and 144 nonaffected horses) to map candidate regions for tying-up syndrome in Japanese Thoroughbreds. The linkage analysis identified one strong L-score (82.45) between the loci UCDEQ411 and COR058 (24.9-27.9 Mb) on ECA12. The GWAS identified two suggestive genomic regions on ECA12 (24.9-27.8 Mb) and ECA20 (29.3-33.5 Mb). Based on both results, the genomic region between UCDEQ411 and TKY499 (24.9-27.8 Mb) on ECA12 was the most significant and was considered as a candidate region for tying-up syndrome in Japanese Thoroughbreds.

A genome-wide association study for racing performances in Thoroughbreds clarifies a candidate region near the MSTN gene.

Using 1400 microsatellites, a genome-wide association study (GWAS) was performed to identify genomic regions associated with lifetime earnings and performance ranks, as determined by the Japan Racing Association (JRA). The minimum heritability (h(2) ) was estimated at 7-8% based on the quantitative trait model, suggesting that the racing performance is heritable. Following GWAS with microsatellites, fine mapping led to identification of three SNPs on ECA18, namely, g.65809482T>C (P=1.05E-18), g.65868604G>T (P=6.47E-17), and g.66539967A>G (P=3.35E-14) associated with these performance measures. The haplotype of these SNPs, together with a recently published nearby SNP, g.66493737C>T (P=9.06E-16) in strong linkage disequilibrium, also showed a very clear association with the performance (P<1E-05). The candidate genomic region contained eight genes annotated by ENSEMBL, including the myostatin gene (MSTN). These findings suggest the presence of a gene affecting the racing performance in Thoroughbred racehorses in this region on ECA18.

Nuclear DISC1 regulates CRE-mediated gene transcription and sleep homeostasis in the fruit fly.

Disrupted-in-schizophrenia-1 (DISC1) is one of major susceptibility factors for a wide range of mental illnesses, including schizophrenia, bipolar disorder, major depression and autism spectrum conditions. DISC1 is located in several subcellular domains, such as the centrosome and the nucleus, and interacts with various proteins, including NudE-like (NUDEL/NDEL1) and activating transcription factor 4 (ATF4)/CREB2. Nevertheless, a role for DISC1 in vivo remains to be elucidated. Therefore, we have generated a Drosophila model for examining normal functions of DISC1 in living organisms. DISC1 transgenic flies with preferential accumulation of exogenous human DISC1 in the nucleus display disturbance in sleep homeostasis, which has been reportedly associated with CREB signaling/CRE-mediated gene transcription. Thus, in mammalian cells, we characterized nuclear DISC1, and identified a subset of nuclear DISC1 that colocalizes with the promyelocytic leukemia (PML) bodies, a nuclear compartment for gene transcription. Furthermore, we identified three functional cis-elements that regulate the nuclear localization of DISC1. We also report that DISC1 interacts with ATF4/CREB2 and a corepressor N-CoR, modulating CRE-mediated gene transcription.

A pilot study to assess the efficacy of photodynamic therapy for Japanese patients with actinic keratosis in relation to lesion size and histological severity.

BACKGROUND/PURPOSE: Topical 5-aminolevulinic acid (ALA)-based photodynamic therapy (PDT) is effective for actinic keratosis (AK); few studies have examined Oriental patients. The aim of this study is to assess the efficacy of PDT for the treatment of Japanese AK patients classified by lesion size and histological severity. METHODS: Thirty patients with solitary AK lesions were divided into two groups according to diameter: a small lesion group (SL), diameter < or =10 mm and a larger lesion group (LL), diameter >10 mm, and histological severity: Group I (mild and moderate) and Group II (severe). After application of 20% ALA for 4 h, exposure to an excimer-dye laser at 630 nm was performed at a dose of 50 J/cm(2) three times at an interval of 7 days. Therapeutic effects were assessed and followed for 12 months. RESULTS: In all 10 SL patients, atypical cells disappeared after PDT and did not recur for 12 months. However, for the 20 LL patients, recurrence was seen in 2 of the 14 Group I patients, while 4 of 6 Group II patients showed residual tumor cells after the first PDT session. CONCLUSION: The present study demonstrated that ALA-PDT might be useful for treatment of Japanese AK. The therapeutic outcome might depend on the lesion size and the histopathological severity.

Etretinate enhances the susceptibility of human skin squamous cell carcinoma cells to 5-aminolaevulic acid-based photodynamic therapy.

BACKGROUND: Photodynamic therapy (PDT) with 5-aminolaevulinic acid (5-ALA) is a noninvasive and effective treatment for superficial skin cancers. Etretinate, a derivate of vitamin A, with the chemical formula ethyl(2E,4E,6E,8E)-9-(4-methoxy-2,3,6-trimethylphenyl)-3,7-dimethyl-2,4,6,8-nona-tetraenoate, has been reported to have antitumour effects and to regulate the proliferation and differentiation of skin cancers. OBJECTIVE: In order to develop more efficient PDT, we investigated whether etretinate enhanced the cytotoxic action of ALA-based PDT against human squamous cell carcinoma cell line, HSC-5. METHOD: The in vitro cytotoxicity was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptotic cells were detected by double-staining with fluorescent annexin V and propidium iodide. Intracellular protoporphyrin IX (PpIX) converted from exogenous ALA was measured by a fluorescence meter. RESULTS: HSC-5 cells pretreated with a nontoxic concentration of etretinate became more susceptible to the cytotoxic action of ALA-based PDT. Etretinate-pretreated cells underwent apoptosis in response to ALA-based PDT. Etretinate pretreatment resulted in enhanced accumulation of ALA-dependent intracellular PpIX. CONCLUSIONS: The results suggest that etretinate enhances the susceptibility of HSC-5 cells to ALA-based PDT via the intracellular increase of ALA-dependent PpIX. Etretinate might be useful for improvement of ALA-based PDT.

Two-stage portal vein ligation facilitates liver regeneration in rats.

BACKGROUND/AIMS: Recent reports have demonstrated that some patients are unable to undergo scheduled liver resection after preoperative portal vein embolization due to insufficient hypertrophy of the future remnant liver. The present study examined whether two-stage portal vein ligation (PVL) increases hypertrophy of the future remnant liver compared to conventional PVL in rats. METHODS: Rats were divided into 3 groups: group A, ligation of left primary branch; group B, ligation of right and left primary branches; group C, ligation of the left primary branch, followed by 2-stage PVL 7 days postoperatively. To evaluate liver regeneration, the proliferating cell nuclear antigen labeling index (LI), mitotic index (MI) in the caudate lobe and weight ratio of caudate lobe to body weight were measured. RESULTS: The weight ratio of caudate lobe to body weight was significantly higher in group C than in groups A or B 14 days postoperatively. In groups A and B, LI and MI in the caudate lobe peaked 2 days postoperatively, then decreased to preoperative levels by 7-8 days postoperatively, but remained significantly elevated until 10-14 days postoperatively in group C. CONCLUSION: Two-stage PVL increases hypertrophy of the future remnant liver compared to conventional PVL in rats.

Memory-like Liver Natural Killer Cells are Responsible for Islet Destruction in Secondary Islet Transplantation.

We previously demonstrated the pivotal role of natural killer (NK) cells in islet graft loss during the early phase after intraportal syngeneic islet transplantation (IT). Liver-resident DX5- NK cells were reported to possess memory-like properties, distinguishing them from conventional DX5+ NK cells. Here, we investigated the impact of primary IT-induced liver DX5- NK cells on the engraftment of secondary-transplanted islets in mice. The culture of liver NK cells isolated from naive mice with TNF-α, IFN-γ, and IL-lß, mimicking instant blood-mediated inflammatory reaction, led to significantly increased DX5- NK cell percentage among total liver NK cells. Consistently, the prolonged expansion of DX5- CD69+ TRAIL+ CXCR3+ NK cells was observed after intraportal IT of 300 syngeneic islets (marginal mass). In most diabetic mice, 400 syngeneic islets of primary IT were sufficient to achieve normoglycaemia, whereas the same mass after secondary IT failed to induce normoglycaemia in mice that received 200 syngeneic islets during primary IT. These findings indicated that liver-resident DX5- NK cells significantly expanded even after syngeneic IT, and that these memory-like NK cells may target both originally engrafted and secondary-transplanted islets. Furthermore, anti-TNF-α treatment suppressed the expansion of liver-resident DX5- NK cells, resulting in successful islet engraftment after sequential ITs.

Effect of tyrosine kinase inhibition by imatinib mesylate on mast cell tumors in dogs.

BACKGROUND: Imatinib mesylate is a small molecule targeted at dysregulated protein-tyrosine kinase. Mutation of c-kit exon 11, which induces constitutive phosphorylation of KIT, is one of the mechanisms for the development or progression of mast cell tumor (MCT) in dogs. The purpose of this study was to examine the therapeutic potential of imatinib mesylate in canine MCT. HYPOTHESIS: Imatinib mesylate has activity against MCT in dogs, and response to treatment can be correlated to presence of mutation within exon 11 of c-kit. ANIMALS: Twenty-one dogs with MCT with gross tumor burden and median tumor size of 7.2 cm (range, 1.0-25.3 cm) before treatment. METHODS: Tumors were analyzed for mutation of c-kit exon 11. Imatinib mesylate was administered PO to the dogs at a dose of 10 mg/kg daily for 1-9 weeks. RESULTS: Ten of 21 dogs (48%) had some beneficial response to imatinib mesylate treatment within 14 days of treatment initiation. All 5 dogs with a demonstrable c-kit mutation in exon 11 responded to the drug (1 complete remission, 4 partial remission). CONCLUSIONS AND CLINICAL IMPORTANCE: Imatinib mesylate has clinical activity against MCT in dogs. Response could not be predicted based on presence of absence of a mutation in exon 11 of c-kit.

Selective elimination of anti-DNA antibody-producing cells by antiidiotypic antibody conjugated with neocarzinostatin.

A new strategy was shown for the manipulation of autoantibody production in humans. Antiidiotypic antibody to human anti-DNA autoantibody was conjugated with neocarzinostatin (NCS), a cytotoxic agent, by using N-succimidyl 3-(2-pyridyldithio) propionate as a coupling agent. Human B cell clones, which produce anti-DNA autoantibodies, were killed by in vitro treatment with antiidiotype (Id)-NCS conjugates, while clones expressing an Id with irrelevant specificity were unaffected. These results indicate that treatment with anti-Id-NCS conjugates can act as a potent and specific means of generating immunosuppression of autoantibody production. This approach will have a significant advantage in aborting clones that are not effectively suppressed for the autoantibodies by anti-Id antibodies alone, and will result in a potential therapeutic treatment for systemic lupus erythematosus.

Cloning and expression of the defective genes from a patient with delta-aminolevulinate dehydratase porphyria.

Cloning and expression of the defective genes for delta-aminolevulinate dehydratase (ALAD) from a patient with inherited ALAD deficiency porphyria (ADP) were carried out. Cloning of cDNAs for the defective ALAD were performed from EBV-transformed lymphoblastoid cells of the proband, and nucleotide sequences were determined. Two separate point mutations resulting in a single amino acid change in each ALAD allele were identified. One, C718----T, termed 'G1', occurred in the allele within the substrate-binding site, producing an Arg240----Trp substitution; the other, G820----A, termed 'G2', occurred downstream of this site in the other allele, resulting in an Ala274----Thr substitution. Using the reverse transcription-polymerase chain reaction, the mother, the brother, and the sister were shown to have the G1 defect. Expression of the G1 cDNA in Chinese hamster ovary cells produced ALAD protein with little activity; the G2 cDNA produced the enzyme with approximately 50% normal activity. Pulse-labeling studies demonstrated that the G1 enzyme had a normal half life, while the G2 enzyme had a markedly decreased half life. These data thus define the separate point mutations in each ALAD allele, as well as the altered properties of the two enzymic proteins encoded by the mutant genes in a patient with ADP.

Nuclear entry mechanism of rat PER2 (rPER2): role of rPER2 in nuclear localization of CRY protein.

Mammalian PERIOD2 protein (PER2) is the product of a clock gene that controls circadian rhythms, because PER2-deficient mice have an arrhythmic phenotype. The nuclear entry regulation of clock gene products is a key step in proper circadian rhythm formation in both Drosophila and mammals, because the periodic transcription of clock genes is controlled by an intracellular, oscillating, negative feedback loop. The present study used deletion mutants of rat PER2 (rPER2) to identify the functional nuclear localization signal (NLS) in rPER2. The elimination of putative NLS (residues 778 to 794) from the rPER2 fragment resulted in the loss of nuclear entry activity. Adding the NLS to the cytosolic protein (bacterial alkaline phosphatase) translocates the fusion protein to the nuclei. The data indicate the presence of a functional NLS in rPER2. Furthermore, intact rPER2 was preferentially translocated from the cytoplasm to the nucleus when coexpressed with human CRY1 (hCRY1). However, rPER2 mutants lacking a carboxyl-terminal domain could not enter the nucleus even in the presence of hCRY1. In addition, coexpression of the nuclear localization domain (residues 512 to 794) lacking rPER2 and CRY1 changed the subcellular localization of CRY1 from the nucleus to the cytoplasm. In vitro protein interaction studies demonstrated that the carboxyl-terminal domain of rPER2 is essential for binding to CRY1. The data suggested that both the rPER2 NLS and carboxyl-terminal CRY binding domain are essential for nuclear entry of the rPER2-CRY1 complex.

Degradation of c-Fos by the 26S proteasome is accelerated by c-Jun and multiple protein kinases.

c-Fos is associated with c-Jun to increase the transcription of a number of target genes and is a nuclear proto-oncoprotein with a very short half-life. This instability of c-Fos may be important in regulation of the normal cell cycle. Here we report a mechanism for degradation of c-Fos. Coexpression of c-Fos and c-Jun in HeLa cells caused marked increase in the instability of c-Fos, whereas v-Fos, the retroviral counterpart of c-Fos, was stable irrespective of the coexpression of c-Jun. Interestingly, deletion of the C-terminal PEST region of c-Fos, which is altered in v-Fos by a frameshift mutation, greatly enhanced its stability, with loss of the effect of c-Jun on its stability. c-Fos synthesized in vitro was degraded by the 26S proteasome in a ubiquitin-dependent fashion. Simple association with c-Jun had no effect on the degradation of c-Fos, but the additions of three protein kinases, mitogen-activated protein kinase, casein kinase II, and CDC2 kinase, resulted in marked acceleration of its degradation by the proteasome-ubiquitin system, though only in the presence of c-Jun. In contrast, v-Fos and c-Fos with a truncated PEST motif were not degraded, suggesting that they escaped from down-regulation by breakdown. These findings indicate a new oncogenic pathway induced by acquisition of intracellular stability of a cell cycle modulatory factor.