Cine MRI Is Useful for the Diagnosis of Intradural Arachnoid Cyst with Spinal Arachnoid Web: A Case Report.

CASE: A 28-year-old woman developed gait disturbance due to lower limb weakness 3 years before presentation. Conventional magnetic resonance imaging (MRI) findings were inconclusive; therefore, we performed cine MRI, which confirmed the presence of a pulsatile cyst on the posterior thoracic spinal cord. The cyst compressed the spinal cord, and its pulsations synchronized with the patient's heartbeats. We resected the intradural arachnoid cyst and thickened arachnoid membrane. The gait disturbance improved after surgery. CONCLUSIONS: Cine MRI can be used to identify a pulsating arachnoid cyst that cannot be visualized with a conventional MRI. Cine MRI is useful in patients with unexplained spinal symptoms.

Formation of taste-active pyroglutamyl peptide ethyl esters in sake by rice koji peptidases.

Formation of taste-active pyroglutamyl (pGlu) peptide ethyl esters in sake was investigated: 2 enzymes (A and B) responsible for the esterification were purified from a rice koji extract. MADLI-TOF/TOF analysis after deglycosylation identified enzyme (A) as peptidase S28 (GenBank accession number OOO13707.1) and enzyme (B) as serine-type carboxypeptidase (accession number AO090010000534). Both enzymes hydrolyzed pGlu peptides and formed ethyl esters under sake mash conditions: acidic pH (3-4) and in ethanol (5%-20% v/v) aqueous solutions. Enzyme (A) formed pGlu penta-peptide ethyl esters from pGlu undeca-peptides by a prolyl endo-type reaction. Enzyme (B) formed (pGlu) deca-peptide and its ethyl esters from pGlu undeca-peptides in an exo-type reaction. We are the first to report the enzymatic ethyl esterification reaction in the formation of pGlu peptides by rice koji peptidases.

Effect of Orthopedics Promotional Activities on Multidisciplinary Management of Patients with Bone Metastases.

BACKGROUND: The rapid aging of the Japanese population is leading to an increase in the number of patients with bone metastases. Since 2014, our orthopedics department has promoted multidisciplinary hospital activities, including offering lectures to hospital staff on multidisciplinary approaches for bone metastases and holding regular cancer board meetings on bone metastases. This study investigated whether these activities were effective in promoting multidisciplinary approaches and improving outcomes of patients with bone metastasis. METHODS: To investigate the effects of changes in medical practice on patients with bone metastases, we compared patient clinical characteristics after (January 2014 through December 2017) and before the start of the activities (January 2011 through December 2013). RESULTS: The semiannual numbers of first-visit, referral, and orthopedic surgical patients, the number of patients with slower growing primary cancers, and the number of patients with milder pain were significantly higher post-activity than pre-activity. The number of patients without paralysis was higher after the start of the activity than before the activity, but the difference was not significant. Survival after the first visit to the orthopedics department was significantly longer after the start of the activity than before the activity. CONCLUSIONS: The potential demand for a multidisciplinary approach to bone metastases is high, and orthopedic specialists should actively participate in this approach.

Factors affecting levels of ferulic acid, ethyl ferulate and taste-active pyroglutamyl peptides in sake.

Factors affecting ferulic acid, ethyl ferulate and taste-active pyroglutamyl (pGlu) peptides levels in sake were analyzed using small-scale sake brewing tests on eighteen rice samples with differing cultivar variety, cropped year and area, and polishing rate. Ferulic acid concentration in sake was highly positively correlated with its content in rice (r = 0.782**, double asterisk indicates 1% significance level), feruloylesterase (FE) activity (r = 0.804**) and feruloylated saccharide forming activity (FSFA) (r = 0.619**) in the rice koji. The results suggested that ferulic acid in rice induced FE activity and FSFA, and these two enzymes accelerated the formation of ferulic acid in sake mash. The concentration of bitter-tasting peptides in sake was highly positively correlated with crude protein content in rice (r = 0.786**), and negatively correlated with acid carboxypeptidase (ACP) activity to (pGlu)LFGPNVNPWH (r = -0.612**), fermentation length (r = -0.820**), and pyroglutamyl leucine ((pGlu)L) concentration in sake (r = -0.502*; 5% significance level). The observation suggested that bitter-tasting peptides are initially formed in sake mash in accordance with protein content in rice, and are then hydrolyzed to smaller peptides, such as (pGlu)L. An ACP specific to the hydrolysis of bitter-tasting peptides was suggested by the observation that ACP activity to (pGlu)LFGPNVNPWH was significantly correlated (-0.612**) to their formation whereas an ACP to Cbz-Glu-Tyr was not (r = 0.220). It was suggested that (pGlu) oligo-peptide ethyl esters were formed during the decomposition of bitter-tasting peptides to which the ACP to (pGlu)LFGPNVNPWH might contribute.

Symptoms Related to Moderate Skeletal-Related Events as Clues for the Diagnosis of Bone Metastasis.

BACKGROUND: Early diagnosis of bone metastasis is difficult. The aim of the present study was to determine whether symptoms related to skeletal-related events (SREs) can be used for the diagnosis of bone metastasis in the absence of screening tests. METHODS: We reviewed 81 patients with bone metastasis to evaluate their SREs at diagnosis. SREs were arbitrarily classified as moderate or severe. Moderate SREs included radiation to the bone before pathological fracture or paralysis, bone surgery before pathological fracture or paralysis, and hypercalcemia without dialysis. Severe SREs included pathological fracture, spinal cord compression, and hypercalcemia necessitating dialysis. RESULTS: The complication rates of SREs at the time the bone metastasis was diagnosed were 59.3% and 24.7% for severe and moderate SREs, respectively, and only 16.0% of cases were uncomplicated. The clinical factors that showed a significant relationship with the severity of SREs were age and history of malignancy. However, there was no significant relationship between the complication rate of total SREs and the presence or absence of a malignancy history (83.3% vs. 85.2%, respectively, p=0.83). CONCLUSION: The results of the present study suggest that symptoms related to SREs can be used to diagnose bone metastasis in the absence of a screening test. Bone metastasis should be diagnosed as often as possible based on symptoms related to moderate SREs and should be treated as soon as possible before patients develop severe SREs.

Development and Verification of Educational Material for Plain Radiographic Diagnosis of Bone Metastasis: A Preliminary Report.

Our previous studies showed that early diagnosis of painful bone metastasis is difficult and requires improvement in the diagnostic accuracy of plain radiography during an initial patient consultation. In this preliminary study, we evaluate the usefulness of educational material used to improve diagnosis of bone metastasis with plain radiography. This study included imaging data from 129 consecutive patients who visited our orthopedic clinic during the period January 2011 through December 2014. First, we prepared a test to measure the reading ability of orthopedic practitioners, after which the educational material was created. Then, the effectiveness of the educational material was verified by having orthopedic trainees take a pre-test and post-test. The test contained plain radiographic data from 12 patients with lesions and 6 without lesions. The educational material included plain radiographic data from 30 patients with typical findings of bone metastasis, as well as diagnostic magnetic resonance images or computed tomography scans, accompanied by a lecture. The accuracy and sensitivity of diagnosis significantly improved after the lecture; however, specificity decreased. Although the educational material was effective for improving the ability of orthopedic trainees to read plain radiographs of bone metastasis, some aspects of the program need to be improved and revised.

Diagnosis of Bone Metastasis in Patients Without a History of Cancer.

BACKGROUND: Diagnosing bone metastasis in patients without a history of cancer remains challenging. Diagnostic evaluation may be prolonged owing to difficulties in distinguishing between bone metastasis and common orthopedic diseases. We hypothesized that bone metastasis due to occult cancer would be more difficult to diagnose than bone metastasis in patients with a history of cancer. Few studies exist on the difficulty of diagnosing bone metastasis in patients without a history of cancer. Therefore, we reviewed the clinical course of patients with bone metastasis between January 2011 and December 2014. METHODS: We reviewed patients with bone metastasis to determine the diagnostic rate at first visit, period from symptom-onset to first visit, period from first visit to diagnosis, and presence of severe skeletal-related events at diagnosis, and compared these between 27 patients without a history of cancer (Group A) and 54 patients with a history of cancer (Group B). RESULTS: The diagnostic rate at first visit was significantly lower (11.5% vs. 52.4%, p=0.00069), the period from first visit to diagnosis was significantly longer (median, 7 weeks vs. 3 weeks, p=0.018), and the presence of severe skeletal-related events at diagnosis was significantly higher (81.4% vs. 50.0%, p=0.05) in patients without a history of cancer compared with those with a history of cancer. CONCLUSIONS: The results of this study indicate that it is difficult to diagnose bone metastasis in patients without a history of cancer. This must be considered in the early diagnosis of bone metastasis to prevent severe skeletal-related events.

Identification of enzymes from genus Trichoderma that can accelerate formation of ferulic acid and ethyl ferulate in collaboration with rice koji enzyme in sake mash.

The enzymes responsible for acceleration of ferulic acid and ethyl ferulate formation in sake mash were studied. Ferulic acid and ethyl ferulate are formed during the sake brewing process from feruloylated glucuronoarabinoxylan. Cellulase reagent from genus Trichoderma was used instead of rice koji, because rice koji for sake brewing produces extremely low levels of xylan-degrading enzymes. A combination of the reagent with rice koji enzymes accelerated the formation of ferulic acid from α-rice powder. Addition of the reagent to sake mash increased ferulic acid and ethyl ferulate formation. The enzyme responsible for the accelerated formation was purified using a newly developed assay method and α-rice powder as a substrate. During the assay procedure, feruloylated oligosaccharide was converted to ferulic acid by feruloylesterase for HPLC analysis. Analysis of the N-terminal amino acid sequence of the purified samples was successfully conducted after pyroglutamyl aminopeptidase de-blocking. Purified enzymes were identified as members of the glycoside hydrolase family 10 (GH10) and family 11 (GH11) xylanases by BLASTP database research. The GH10 xylanase showed higher specific activity for α-rice powder and insoluble wheat arabinoxylan compared with GH11 xylanase; the GH11 xylanase showed higher specific activity for the other xylan substrates, especially glucuronoarabinoxylan. The GH10 xylanase showed higher accelerating activity than the GH11 xylanase in the sake mash. The results of this study provides useful knowledge on ferulic acid and ethyl ferulate formation in sake mash, the relative levels of these compounds and their influence on the sensory quality of sake.

Quantitation and sensory properties of three newly identified pyroglutamyl oligopeptides in sake.

Three new peptides: (pGlu)L-ethyl, (pGlu)LFGP-ethyl and (pGlu)LFNP-ethyl, were identified in the search for pyroglutamyl oligopeptide ethyl esters in sake. The ethyl esterified peptides in sake were quantitated using stable isotope dilution analysis and additional quantitation of (pGlu)L was performed using an external standard method. The concentrations of (pGlu)L-ethyl and (pGlu)L in 33 commercial sake samples ranged from 0.16 to 1.57 mg/L and 1.49 to 7.55 mg/L, respectively. The sensory properties of the pyroglutamyl oligopeptide ethyl esters and corresponding non-esterified peptides were examined: the estimated difference threshold of (pGlu)L (2.0 mg/L) and (pGlu)L-ethyl (0.267 mg/L) was exceeded in 32 and 26 samples, respectively. Estimated thresholds of (pGlu)LFGP-ethyl and (pGlu)LFNP-ethyl were often lower than the levels in quantitated sake samples. The sensory effects of these pyroglutamyl dipeptides on a model sake quality may be negative because of their unpleasant taste, however, (pGlu)LFNP-ethyl may be positive because of its mild taste.

Diagnostic Value of Plain Radiography for Symptomatic Bone Metastasis at the First Visit.

BACKGROUND: To prevent and minimize skeletal-related diseases, early diagnosis of bone metastases is important. However, previous reports have shown that plain radiography has low sensitivity and fails to screen multiple asymptomatic lesions. Limited investigations have been reported on the value of plain radiography in the diagnosis of symptomatic bone metastases. Therefore, this study aimed to investigate the diagnostic utility of plain radiography for symptomatic bone metastasis. METHODS: Two experienced orthopedic surgeons retrospectively evaluated the plain radiographs of 39 patients with symptoms during their first visit between 2011 and 2014 for bone metastases. Another 2 experienced orthopedic surgeons then reviewed the data using 2 reference standards, the clinical results and the retrospectively evaluated results, in a blinded manner. The data were then reviewed by 2 certified orthopedic surgeons and 7 orthopedic surgeons in training with differing years of experience in a blinded manner. RESULTS: The overall sensitivity of diagnosis of symptomatic bone metastasis using plain radiography at the clinic first visit was 71.4%. Upon blinded evaluation, the accuracy, sensitivity, and specificity were 55.8%, 54.3%, and 68.8% and 77.6%, 73.0%, and 85.7% for clinical results and results from 2 experienced orthopedic surgeons as a reference standard, retrospectively. There was a strong and significant correlation between the accuracy and observers' years of experience in orthopedic surgery among the orthopedic surgeons in training (R=0.942, p=0.0015). CONCLUSIONS: Plain radiography around the time of the first visit has a definitive role in the early diagnosis of symptomatic bone metastasis.

Challenges in the Diagnosis of bone Metastasis in Patients without a History of Malignancy at Their First Clinic Visit.

BACKGROUND: Many patients with bone metastases whose diagnoses came only after they had suffered aggravated conditions are still frequently encountered. However, there have been few studies regarding the early diagnosis of such metastases. We retrospectively reviewed the clinical courses of cases we experienced between 2004 and 2014 to clarify the practical situation of diagnosis of such bone metastases. METHODS: We undertook a retrospective review of 56 of our patients with bone metastasis who had no history of malignancy at their first visit, who visited our departments between 2004 and 2014. The initial diagnoses at the first visit to any clinic, the period from the first visit to any clinic to diagnosis of bone metastasis, the process to make the diagnosis, the frequency of severe skeletal-related events at diagnosis, and the examination serving as the basis for diagnosis were evaluated. RESULTS: The diagnosis of bone metastasis was made at the first visit in only 6 of the 56 patients. Pathological fractures, paralysis and/or calcemia were seen in 62.5% of the patients at diagnosis of bone metastasis. The median period from the first visit to any clinic to diagnosis was 7.0 weeks. Typically, the diagnosis of bone metastasis was made only after aggravation. The most frequent examination to serve as the basis of diagnosis was magnetic resonance imaging. CONCLUSIONS: Diagnosis of bone metastasis is challenging in patients without a history of malignancy at their first visit. For early diagnosis, it is important to recognize this challenge and to keep it in mind together with ongoing observation.

Development of diabetes mellitus associated with quetiapine: A case series.

We aimed to describe the characteristics and clinical course of patients who developed diabetes associated with the use of quetiapine.This study included patients who received quetiapine for over a month between April 2008 and November 2013, and were diagnosed as having new-onset diabetes after initiation of quetiapine. We excluded patients who developed diabetes more than 1 year after discontinuation of quetiapine. We identified new-onset diabetes by hemoglobin A1c or prescriptions of antidiabetic drugs.Among 1688 patients who received quetiapine, hemoglobin A1c had been measured in 595 (35.2%) patients at least once during the observation period, and 33 (2.0%) patients had received hypoglycemic drugs. Eighteen (1.1%) patients were considered to have developed new-onset diabetes associated with quetiapine after a median of 1.6 years following initiation of quetiapine. Median (interquartile range) age was 54.5 (29.8) years, 8 patients were male, and median (interquartile range) duration of mental illness was 15.3 (13.8) years. Median hemoglobin A1c and body mass index (BMI) were 7.1 (1.4) % and 28.4 (7.0) kg/m, respectively. Seventeen patients had dyslipidemia when diabetes was discovered. All of these discontinued quetiapine within 3 months after the diagnosis of diabetes, and the diabetes in 4 patients had ameliorated without hypoglycemic drugs. Of 13 patients who had received either oral hypoglycemic drugs or insulin, 2 patients achieved well-controlled hemoglobin A1c without hypoglycemic drugs, and 10 patients had hemoglobin A1c 5.0% to 7.7% with the continued use of hypoglycemic drugs.We demonstrated that almost all patients who developed quetiapine-associated diabetes had dyslipidemia and increased BMI. There was no life-threatening hyperglycemia and diabetes was ameliorated just by discontinuation of quetiapine in several patients. The monitoring of metabolic parameters during antipsychotic treatment is important to diagnose and treat diabetes earlier.

Formation of Guaiacol by Spoilage Bacteria from Vanillic Acid, a Product of Rice Koji Cultivation, in Japanese Sake Brewing.

The formation of guaiacol, a potent phenolic off-odor compound in the Japanese sake brewing process, was investigated. Eight rice koji samples were analyzed, and one contained guaiacol and 4-vinylguaiacol (4-VG) at extraordinarily high levels: 374 and 2433 µg/kg dry mass koji, respectively. All samples contained ferulic and vanillic acids at concentrations of mg/kg dry mass koji. Guaiacol forming microorganisms were isolated from four rice koji samples. They were identified as Bacillus subtilis, B. amyloliquefaciens/subtilis, and Staphylococcus gallinarum using 16S rRNA gene sequence. These spoilage bacteria convert vanillic acid to guaiacol and ferulic acid to 4-VG. However, they convert very little ferulic acid or 4-VG to guaiacol. Nine strains of koji fungi tested produced vanillic acid at the mg/kg dry mass koji level after cultivation. These results indicated that spoilage bacteria form guaiacol from vanillic acid, which is a product of koji cultivation in the sake brewing process.

Formation of ethyl ferulate from feruloylated oligosaccharide by transesterification of rice koji enzyme under sake mash conditions.

Formation of ethyl ferulate (EF) and ferulic acid (FA) under sake mash conditions was studied using feruloylated oligosaccharide (FO), prepared from rice grains, as the substrate for rice koji enzyme. EF and FA were produced from FO over six times faster than from alkyl ferulates however, under the same ethanol concentration, only small differences were observed between the EF/FA ratios when either FO or methyl ferulate were used as substrates. Esterification and hydrolysis of FO or methyl ferulate showed similar pH dependencies and similar EF/FA ratios for each substrate in all of the pH ranges tested. Ethanol concentration clearly affected the EF/FA ratio; the ratio increased as ethanol concentration increased. Formation of EF and FA in the sake mash simulated rice digest was accelerated by addition of exogenous FO. These results indicated that supply of FO to sake mash is a crucial step for EF and FA formation, and ethanol is an influencing factor in the EF/FA ratio. The rice koji enzyme reaction suggested that EF and FA are formed through a common feruloylated enzyme intermediate complex by transesterification or hydrolysis, and these reactions occur competitively.

Generation of aminoterminally truncated, stable types of bioactive bovine and porcine fibroblast growth factor 4 in Escherichia coli.

Fibroblast growth factor 4 (FGF4) is a crucial growth factor for the development of mammalian embryos. We previously produced hexahistidine-tagged, bovine and porcine FGF4 (Pro(32) to Leu(206) ) proteins without a secretory signal peptide at the aminoterminus in Escherichia coli. Here, we found that these were unstable; site-specific cleavage between Ser(54) and Leu(55) in both FGF4 derivatives was identified. In order to generate stable FGF4 derivatives and to investigate their biological activities, aminoterminally truncated and hexahistidine-tagged bovine and porcine FGF4 (Leu(55) to Leu(206) ) proteins, termed HisbFGF4L and HispFGF4L, respectively, were produced in E. coli. These FGF4 derivatives were sufficiently stable and exerted mitogenic activities in fibroblasts. Treatment with the FGF4 derivatives promoted the phosphorylation of ERK1/2, which are crucial kinases in the FGF signaling pathway. In the presence of PD173074, an FGF receptor inhibitor, the phosphorylation of ERK1/2 was inhibited and resulted in abolition of the growth-promoting activity of FGF4 derivatives. Taken together, we demonstrate that HisbFGF4L and HispFGF4L are capable of promoting the proliferation of bovine- and porcine-derived cells, respectively, via an authentic FGF signaling pathway. These FGF4 derivatives may be applicable for dissecting the roles of FGF4 during embryogenesis in cattle and pigs.

Mechanism of high trehalose accumulation in a spore clone isolated from Shirakami kodama yeast.

The intracellular trehalose levels in Shirakami kodama yeast, a strain of Saccharomyces cerevisiae, isolated in 1997 from leaf mold in the Shirakami Mountains and since used as a commercial baker's yeast, are remarkably high, which presumably is related to its tolerance of freezing and drought conditions. We isolated a spore clone from Shirakami kodama yeast with about 1.7-fold higher intracellular trehalose levels than the parental strain and set out to elucidate how this spore clone can accumulate intracellular trehalose to such a high concentration. The gene for trehalose 6-phosphate synthase, TPS1, was duplicated in this spore clone. Both TPS1 genes contributed to the high level of intracellular trehalose as a 3.4-fold decrease resulted from the disruption of one of the two TPS1 genes. Both Msn2 and Msn4, which bind to stress responsive elements in the promoter region of TPS1, were required for production of high levels of trehalose. Furthermore, the neutral trehalase activity of this spore clone is about 3-fold less than that of the laboratory strain although the gene for neutral trehalase, NTH1, functioned normally. These findings indicate that two TPS1 genes and the low trehalase activity are associated with high trehalose accumulation in this spore clone. The wide range of stresses of which we found the spore clone to be tolerant makes this yeast very attractive for commercial application and for further research into the mechanisms underlying stress responses and trehalose metabolism.

Factors affecting phenolic acid liberation from rice grains in the sake brewing process.

Phenolic acid (ferulic and p-coumaric acid) liberation from rice grains was examined using rice samples containing phenolic acid at different levels, using two sake mash simulated digestion tests to elucidate influencing factors. Phenolic acid levels in a digest made from steamed rice using dialyzed rice koji enzymes were smaller than levels in a rice koji self-digest. Differences in phenolic acid levels among rice samples in the rice koji self-digest were larger than levels in a digest of steamed rice. In the rice koji self-digest, phenolic acid levels in the ingredient rice grains or in the formed digest related to feruloylesterase (FE) activity in the rice koji. Addition of exogenous FE to rice koji self-digestion increased phenolic acid levels, while addition of xylanase (Xyl) showed weak effects. A concerted effect of FE and Xyl was not clearly observed. Addition of ferulic acid to koji made from α-rice grains raised FE activity, but it did not increase the activity of other enzymes. A similar phenomenon was observed in an agar plate culture of koji mold. These results indicated that ferulic acid levels in ingredient rice grains correlate with FE activities of koji, as a resulut, they affect the phenolic acid levels in sake mash.

Production of an aminoterminally truncated, stable type of bioactive mouse fibroblast growth factor 4 in Escherichia coli.

In mice, fibroblast growth factor 4 (Fgf4) is a crucial gene for the generation of trophectoderm, progenitor cells of the placenta. Therefore, exogenous FGF4 promotes the isolation and maintenance of trophoblast stem cells from preimplantation embryos. We previously produced a 6× histidine (His)-tagged, mouse FGF4 (Pro(31)-Leu(202)) without a secretory signal peptide at the amino-terminus, referred to as HismFGF4, in Escherichia coli. Here, we found that HismFGF4 was unstable, such as in phosphate-buffered saline. In these conditions, site-specific cleavage between Ser(50) and Leu(51) was identified. In order to generate stable mouse FGF4 derivatives, a 6× His-tagged mouse FGF4 (Leu(51)-Leu(202)), termed HismFGF4L, was expressed in E. coli. HismFGF4L could be purified from the supernatant of cell lysates by heparin column chromatography. In phosphate-buffered saline, HismFGF4L was relatively stable. HismFGF4L exerted significant mitogenic activities at concentrations as low as 0.01 nM (P < 0.01) in mouse embryonic fibroblast Balb/c 3T3 cells expressing FGF receptor 2. In the presence of PD173074, an FGF receptor inhibitor, the growth-promoting activity of HismFGF4L was abolished. Taken together, we suggest that aminoterminally truncated HismFGF4L is capable of promoting the proliferation of mouse-derived cells via an authentic FGF signaling pathway. We consider that HismFGF4L is useful as a derivative of mouse FGF4 protein for analyzing the effects of mouse FGF4 and for stimulating cell growth of mouse-derived cells, such as trophoblast stem cells. Our study provides a simple method for the production of a bioactive, stable mouse FGF4 derivative in E. coli.

Identification of site-specific degradation in bacterially expressed human fibroblast growth factor 4 and generation of an aminoterminally truncated, stable form.

Fibroblast growth factor 4 (FGF4) is considered as a crucial gene for tumorigenesis in humans and the development of mammalian embryos. The secreted, mature form of human FGF4 is thought to be comprised of 175 amino acid residues (proline(32) to leucine(206), Pro(32)-Leu(206)). Here, we found that bacterially expressed, 6× histidine (His)-tagged human FGF4 (Pro(32)-Leu(206)) protein, referred to as HishFGF4, was unstable such as in phosphate-buffered saline. In these conditions, site-specific cleavage, including between Ser(54) and Leu(55), in HishFGF4 was identified. In order to generate stable human FGF4 derivatives, a 6× His-tagged human FGF4 (Leu(55)-Leu(206)), termed HishFGF4L, was expressed in Escherichia coli. HishFGF4L could be purified from the supernatant of cell lysates by heparin column chromatography. In phosphate-buffered saline, HishFGF4L was considered as sufficiently stable. HishFGF4L exerted significant mitogenic activities in mouse embryonic fibroblast Balb/c 3T3 cells. In the presence of PD173074, an FGF receptor inhibitor, the growth-stimulating activity of HishFGF4L disappeared. Taken together, we suggest that HishFGF4L is capable of promoting cell growth via an authentic FGF signaling pathway. Our study provides a simple method for the production of a bioactive human FGF4 derivative in E. coli.

[Clinical experience with clozapine in 55 cases of treatment-resistant schizophrenia].

Up until October 2012, Kohnodai Hospital had introduced clozapine treatment for 55 cases of treatment-resistant schizophrenia. In all cases, previous antipsychotic medication was discontinued the day before clozapine administration began. Of the 55 cases, 45(85%)are continuing clozapine administration, and 40 cases (73%) are receiving outpatient treatment. The average dose of clozapine was 373.1 mg/day (SD : 160.5). Clozapine was administered for a month or more in 51 cases (93%). BPRS scores improved 20% or more in a month's administration of clozapine in 18 of the cases (35%). The average clozapine dose in the improvement cases was 176 mg/day. The average BPRS score had significantly decreased from the baseline at months 1, 3, 6, and 12 after the start of clozapine administration. Of the 33 cases receiving clozapine treatment for 12 months or more, BPRS improved 20% or more in 27 (82%). BPRS improved 20% or more for the first time after clozapine administration within a month in 12 cases (44%), 3 months in 8 cases (30%), 6 months in 5 cases (19%), and 12 months in 2 cases (7%). These results suggest that clozapine should be administered continuously for over 6 months at the least and 12 months if possible to evaluate the efficacy of clozapine treatment. Of the 43 cases receiving outpatient clozapine therapy, the average GAF score improved significantly from the time of ward admission to discharge (20.6 and 42.0, respectively). Clozapine had to be discontinued in 2 cases of leukopenia, 2 cases of neutropenia, 1 case of reduced left ventricular ejection due to pericardial effusion, 1 case of drug eruption, and 1 case of marked hunger. When introducing clozapine for treatment-resistant schizophrenia, it is important to administer it as a monotherapy, slowly increase the dosage to reduce side effects, and achieve a treatment effect at the minimum required dosage.