Search

Aims Coronary arteritis is a life-threatening complication that may arise in the acute stage of Kawasaki disease (KD), the leading cause of systemic vasculitis in childhood. Various microorganisms and molecular pathogens have been reported to cause KD. However, little is known about the key molecules that contribute to the development of coronary arteritis in KD. Methods and results To identify causative molecules for coronary arteritis in KD, we prospectively recruited 105 patients with KD and 65 disease controls in four different parts of Japan from 2015 to 2018. During this period, we conducted lipidomics analyses of their sera using liquid chromatography–mass spectrometry (LC-MS). The comprehensive LC-MS system detected a total of 27 776 molecules harbouring the unique retention time and m/z values. In the first cohort of 57 KD patients, we found that a fraction of these molecules showed enrichment patterns that varied with the sampling region and season. Among them, 28 molecules were recurrently identified in KD patients but not in controls. The second and third cohorts of 48 more patients with KD revealed that these molecules were correlated with inflammatory markers (leucocyte counts and C-reactive proteins) in the acute stage. Notably, two of these molecules (m/z values: 822.55 and 834.59) were significantly associated with the development of coronary arteritis in the acute stage of KD. Their fragmentation patterns in the tandem MS/MS analysis were consistent with those of oxidized phosphatidylcholines (PCs). Further LC-MS/MS analysis supported the concept that reactive oxygen species caused the non-selective oxidization of PCs in KD patients. In addition, the concentrations of LOX-1 ligand containing apolipoprotein B in the plasma of KD patients were significantly higher than in controls. Conclusion These data suggest that inflammatory signals activated by oxidized phospholipids are involved in the pathogenesis of coronary arteritis in KD. Because the present study recruited only Japanese patients, further examinations are required to determine whether oxidized PCs might be useful biomarkers for the development of coronary arteritis in broad populations of KD.

Background The role of therapeutic intervention, particularly antibiotics, for Shiga toxin-producing Escherichia coli (STEC) related infection is controversial. Methods We performed a population based matched case-control study to assess the association between treatment (antibiotics, antidiarrheal agents and probiotics) for STEC related infections and HUS development. We identified all STEC HUS patients as cases and matched five non-HUS patients as controls using the data from the National Epidemiological Surveillance of Infectious Diseases (NESID) between January 1, 2017, and December 31, 2018. Further medical information was obtained by standardized questionnaires answered by physicians who registered each patient. We used multivariate conditional logistic regression model to evaluate the association between exposures (use of antibiotics, use of antidiarrheal agents, days between disease onset and fosfomycin administration [within two or three days]) and the development of HUS, by matched odds ratios (OR) and 95% confidence intervals (CI). Covariates we used were sex, age group, area code, presence of diarrhea and other factors. We also performed subgroup analyses using age (adults and children) as a stratification factor. Results 7,760 STEC related patients were registered in the NESID. We selected patients who had a record of HUS diagnosis (n=182) and matched controls without HUS (n=910). After collecting standardized paper-based questionnaires, we enrolled 90 HUS patients and 371 non-HUS patients for analysis. In the main analysis, matched OR of fosfomycin was 0.75(0.47-1.20) in all ages, 1.41(0.51-3.88) in adults and 0.58(0.34-1.01) in children. Matched OR of antidiarrheal agents was 2.07(1.07-4.03) in all ages, 1.84(0.32-10.53) in adults, 2.65(1.21-5.82) in children. Matched OR of probiotics was 0.86(0.46-1.61) in all ages, 0.76(0.21-2.71) in adults, 1.00(0.48-2.09) in children. There was no significant association between the timing of fosfomycin use in the first two or five days of illness and HUS development in any age group. Conclusion Our results suggest that fosfomycin might decrease the risk of HUS in children younger than 15 years of age with STEC confirmed bacterial gastroenteritis. Disclosures All Authors: No reported disclosures

An alkaline κ-carrageenase, Cgk-K142, was found in the culture broth of a deep-sea bacterium, Pseudoalteromonas tetraodonis JAM-K142. A gene for the enzyme was cloned and expressed. Purified recombinant Cgk-K142 (rCgk-K142) showed an optimal pH of about 8.8 in glycine-NaOH buffer at 30 °C and of about 8.0 in MOPS buffer at 50 °C. The optimal temperature for the enzyme was 55 °C at pH 8.0. rCgk-K142 was unstable, but λ- and ι-carrageenans, non-degradative substrate homologs, extensively enhanced its stability. The nucleotide sequence of the gene for Cgk-K142 comprised 1,194 bp, and the deduced amino acid sequence (397 amino acids) showed a high level of similarity to the κ-carrageenase of P. carrageenovora, with 94% identity. Another gene for a κ-carrageenase-like protein was found downstream of the gene for Cgk-K142. The nucleotide sequence of that gene consisted of 966 bp (321 amino acids), and it showed the highest similarity, at 64% identity, to protein CgkB of P. carrageenovora, which has been reported as an incomplete 57-amino acid sequence.

The natural clonal loach Misgurnus anguillicaudatus (Teleostei: Cobitidae) is diploid (2n = 50) and produces genetically identical unreduced eggs, which develop into diploid individuals without any genetic contribution from sperm. Artificially sex-reversed clones created by the administration of 17alpha-methyltestosterone produce clonal diploid sperm. In metaphase spreads from testicular cells of the sex-reversed clones, spermatocytes had twice the normal number of chromosomes (50 bivalents) compared with those of normal diploids (25 bivalents). Thus, the production of unreduced diploid spermatozoa is initiated by premeiotic endomitosis (or endoreduplication), chromosome doubling before meiosis, and is followed by two quasinormal divisions. Larger nuclei in the germ cells were observed in all stages of type B spermatogonia in the testes of the sex-reversed clones. In contrast, besides having larger type A spermatogonia, the sex-reversed clones also had the type A spermatogonia that were the same size as those of normal diploids. It follows that chromosome duplication causing unreduced spermatogenesis occurred in the type A spermatogonia. The presence of tetraploid type A and early type B spermatogonia, identified by labeling with antispermatogonia-specific antigen 1, was verified using DNA content flow cytometry. These results support the conclusion that chromosome doubling occurs at the type A spermatogonial stage in diploid spermatogenesis in the clonal fish.

The Nile tilapia, a gonochoristic teleost fish with an XX/XY sex-determining system, provides an excellent model for studying gonadal sex differentiation because genetic all-females and all-males are available. In this study, we used quantitative real-time RT-PCR to determine the precise timing of the gonadal expression of 17 genes thought to be associated with gonadal sex differentiation in vertebrates. Gonads were isolated from allfemale and all-male tilapia before (5–15 days after hatching [dah]) and after (25–70 dah) morphological sex differentiation. The transcript of aromatase (cyp19a1a), an enzyme responsible for producing estradiol-17beta, was expressed only in XX gonads at 5 dah, with a marked elevation in expression thereafter. In contrast, mRNA expression of steroid 11beta-hydroxylase (cyp11b2), an enzyme responsible for the synthesis of 11ketotestosterone (11-KT, a potent androgen in fish), was found in XY gonads from 35 dah only. These results, combined with the presence of transcripts for other steroidogenic enzymes and estrogen receptors in XX gonads at 5–7 dah, are consistent with our earlier suggestion that estradiol-17beta plays a critical role in ovarian differentiation in tilapia, whereas a role for 11-KT in testicular differentiation is questionable. A close relationship between the expression of foxl2, but not nr5a1 (Ad4BP/SF-1), and that of cyp19a1a in XX gonads suggests an important role for Foxl2 in the transcriptional regulation of cyp19a1a. Dmrt1 exhibited a male-specific expression in XY gonads from 6 dah onward, suggesting an important role for Dmrt1 in testicular differentiation. Sox9 and amh (anti-Mullerian hormone) showed a testis-specific expression, being evident only in the later stages of testicular differentiation. It is concluded that the sex-specific expression of foxl2 and cyp19a1a in XX gonads and dmrt1 in XY gonads during early gonadal differentiation (5–6 dah) is critical for undifferentiated gonads to differentiate into either the ovary or testis in the Nile tilapia. aromatase, DMRT1, fish, Foxl2, sex differentiation

Involvement of Ad4BP/SF-1 in the ovarian cytochrome P-450 aromatase (oP450arom) gene expression was investigated using ovarian follicles of the Nile tilapia, possessing an average 14day spawning cycle. The promoter region (59 flanking region) of oP450arom gene cloned from tilapia contains two Ad4 binding sites. Subsequently, a cDNA encoding Ad4BP/SF-1 was cloned from the ovarian follicles. It is expressed in gonadal tissues, brain, and kidney. Oligonucleotide probes containing putative orphan nuclear receptor binding motifs (derived from promoter region of the aromatase gene) formed complexes with in vitrotranslated Ad4BP/SF-1 and nuclear extracts of tilapia ovarian (midvitellogenic) follicles, indicating that Ad4BP/SF-1 is one of the transcriptional regulators for aromatase gene expression. Northern blot analysis revealed that the expression of both oP450arom and Ad4BP/SF-1 increased in parallel with ovarian growth from Day 0 to Day 5 after spawning and declined sharply from Day 8 to Day 11. On the day of spawning (Day 14), the expression of both correlates became undetectable. In vitro incubation of post vitellogenic full-grown immature follicles (corresponding to Day 11 after spawning) with hCG purged both oP450arom and Ad4BP/SF-1 messenger RNA transcripts at 18 h. Conversely, in vitro incubation of late vitellogenic follicles (corresponding to Day 8 after spawning) with hCG retained Ad4BP/ SF-1 messenger RNA transcripts more or less steadily and upregulated oP450arom. Ad4BP/SF-1 probably acts as a transcriptional modulator to implement the paradoxical actions of gonadotropins on oP450arom gene. follicular development, granulosa cells, luteinizing hormone, ovulation, steroid hormones

Meiotic maturation in fish is accomplished by maturationinducing hormones. 17a,20b-Dihydroxy-4-pregnen-3-one (17a,20b-DP) was identified as the maturation-inducing hormone of several teleosts, including Nile tilapia. A cDNA encoding 20b-hydroxysteroid dehydrogenase (20b-HSD), the enzyme that converts 17a-hydroxyprogesterone to 17a,20b-DP, was cloned from the ovarian follicle of Nile tilapia. Genomic Southern analysis indicated that 20b-HSD probably exists as a single copy in the genome. The Escherichia coli-expressed cDNA product oxidized both carbonyl and steroid compounds, including progestogens, in the presence of NADPH. Carbonyl reductase-like 20b-HSD is broadly expressed in various tissues of tilapia, including ovary, testis, and gill. Northern blot and reverse transcription polymerase chain reaction analyses during the 14-day spawning cycle revealed that the expression of 20b-HSD in ovarian follicles is low from Day 0 to Day 8 after spawning and is not detectable on Day 11. Distinct expression was evident at Day 14, the day of spawning. In males, 20bHSD expression was observed continually in mature testes but not in immature testes of 30-day-old fish. In vitro incubation of postvitellogenic immature follicles (corresponding to Day 11 after spawning) with hCG induced the expression of 20bHSD mRNA transcripts within 1‐2 h, followed by the final meiotic maturation of oocytes. In tissues such as gill, muscle, brain, and pituitary, however, hCG treatment did not induce any changes in the levels of mRNA transcripts. Actinomycin D blockade of hCG-induced 20b-HSD expression and final oocyte maturation demonstrated the involvement of transcriptional factors. The carbonyl reductase-like 20b-HSD plays an important role in the meiotic maturation of tilapia gametes. gametogenesis, granulosa cells, ovary, ovulation, steroid hormones

An alkaliphilic Bacillus sp. strain, KSM-64, produces a mesophilic alkaline endo-1,4-beta-glucanase that is suitable for use in detergents. The deduced amino acid sequence of the enzyme showed very high homology to that of a thermostable alkaline enzyme from alkaliphilic Bacillus sp. strain KSM-S237. Analysis of chimeric enzymes produced from the genes encoding the mesophilic and thermostable enzymes suggested that the lysine residues at positions 137, 179, and 194 are responsible for their thermal stabilization. Replacing the corresponding Glu137, Asn179, and/or Asp194 with lysine by site-directed mutagenesis made the mesophilic enzyme more thermostable. Analyses of the hydrophilicity of deduced amino acid sequences and isoelectric focusing of the modified enzymes suggested that these three specific lysine residues and their replacements are all located on the surface of the enzyme molecule. This fact further suggested that specific ionic interaction is involved in the thermal stabilization of the enzyme.

Alkaliphilic Bacillus sp. strain KSM-S237 (a relative of Bacillus pseudofirmus) produces a thermostable, alkaline endo-1,4-beta-glucanase (Egl). The entire gene for the enzyme harbored a 2,472-bp open reading frame (ORF) encoding 824 amino acids, including a 30-aminoacid signal peptide. The deduced amino acid sequence of the mature enzyme (794 amino acids, 88,284 Da) showed very high similarity to those of family 5 mesophilic, alkaline Egls from some alkaliphilic bacilli. The enzyme had a region similar to a novel cellulose binding domain proposed for an Egl (EngF) from Clostridium cellulovorans. Expression of the Bacillus Egl gene in Bacillus subtilis resulted in high carboxymethy cellulase activity (2.0 g/l) in the culture broth, concomitant with the appearance of a protein band on an SDS gel at 86 kDa. Site-directed mutagenesis delineated the importance of Arg111, His151, Glu190, His262, Tyr264, and Glu305 in catalysis and/or substrate binding of the enzyme.

Background Patients with acute lymphocytic leukemia (ALL) and those with lymphoblastic lymphoma (LBL) have overlapping clinical and immunophenotypic features and they have been treated with the same or very similar chemotherapy regimens. The goal of this multi-institutional phase II trial was to evaluate the therapeutic efficacy of a short-term, six-drug chemotherapy regimen for adult patients with untreated ALL or LBL. Methods Forty-six eligible patients, 41 with ALL and five with LBL, were treated with a short-term (planned total therapy duration; 36-38 weeks), simplified chemotherapy program; two courses of VEPA-L (vincristine, cyclophosphamide, prednisolone, doxorubicin, I-asparaginase plus intrathecal methotrexate and prednisolone) followed by four courses of M-VEPA (methotrexate plus VEPA), without the traditional maintenance therapy using daily 6-mercaptopurine and weekly methotrexate. Results Thirty-six (78%; 95% confidence interval 64-89%) of the 46 eligible patients achieved complete remission (CR). Among the 36 patients who achieved CR, four (11%) died of treatment complications, 26 (72%) relapsed and six (17%) remain alive in continuous CR. The median survival for all 46 eligible patients is 14 months and the median disease-free survival (DFS) for the 36 patients who achieved CR is 11 months. The estimate of the proportion of survival at 7 years of all 46 eligible patients is 15% at a median follow-up time of 96 months and that of DFS of the 36 patients achieving CR is 17% at a median follow-up time of 93 months. Subgroup analysis showed that an elevated serum C-reactive protein (CRP) level, age of 30 years or older, the presence of B-symptom and T-cell phenotype were likely to be associated with shortened survival. Although the observed CR rate (78%) is within the range of satisfaction, the long-term survival rate (15%) is inferior to those of published programs incorporating maintenance therapy. Conclusions A fraction of adult patients with ALL or LBL are curable with a short-term, six-drug chemotherapy regimen. However, this simplified therapy of shorter duration cannot be recommended.

A low-molecular-weight, high-alkaline pectate lyase (pectate transeliminase, EC 4.2.2.2) was found in an alkaline culture of Bacillus sp. strain KSM-P15, purified to homogeneity, and crystallized. The enzyme had a relative molecular weight of approximately 20,300 as measured by sedimentation equilibrium, with a sedimentation coefficient (s20,w0) of 1.73 S. It was a basic protein with an isoelectric point of pH 10.3, and the alpha-helical content was only 6.6%. In the presence of Ca2+ ions, the enzyme degraded polygalacturonic acid in a random manner to yield 4,5-unsaturated oligo-galacturonides and had its optimal activity around pH 10.5 and 50-55 degrees C. It also had a protopectinase-like activity on cotton fibers. The N-terminal amino acid sequences of the intact protein (28 amino acids) and its two lysyl endopeptidase-cleaved peptide fragments (8 and 12 amino acids) had very low sequence similarity with pectate lyases reported to date. These results strongly suggest that the pectate lyase of Bacillus sp. strain KSM-P15 may be a novel enzyme and belongs in a new family.

Irinotecan hydrochloride (CPT-11) is a new derivative of camptothecin which inhibits topoisomerase I. Phase II studies have demonstrated that CPT-11 is active against a broad spectrum of neoplasms including intractable non-Hodgkin's lymphoma. An early phase II study in lymphoma suggested that a schedule of daily infusions of 40 mg/m2/day for three or five consecutive days is more effective than a single infusion of 200 mg/m2 every three to four weeks. Carboplatin is also an active agent against lymphoma, and preclinical studies have shown that CPT-11 and its active metabolite have a synergistic effect with platinum compounds. To evaluate the maximal tolerated dose (MTD) and the therapeutic efficacy of CPT-11 in combination with carboplatin in relapsed or refractory non-Hodgkin's lymphoma, we conducted a combination phase I/II study. The starting dose of CPT-11 was 20 mg/m2/day (days 1 through 3 and 8 through 10), and dose escalations of 5 mg/m2/day increments were planned, with a fixed dose of carboplatin (300 mg/m2, day 1). Six of the eight patients receiving both agents at the starting dose level developed critical toxicities such as grade 4 hematologic (neutropenia 6/8, thrombocytopenia 1/8) and grade 3 non-hematologic toxicities (diarrhea 2/8, transaminase elevation 1/8). Further dose escalation of CPT-11 was halted, and the starting doses were judged to be the MTDs. The response rate (25%, 2/8) to the combination of the MTDs was not superior to that of CPT-11 alone in a previous phase II study (38%, 26/69), and the MTD of CPT-11 in combination with carboplatin was less than half the single-agent dose. We conclude that carboplatin is not recommendable for combination with CPT-11 in lymphoma patients. Other suitable agents for such a combination should be sought.

In Nile tilapia, sex-specific expression of foxl2 and cyp19a1a in XX gonads and dmrt1 in XY gonads at 5–6 days after hatching (dah) is critical for differentiation of the gonads into either ovaries or testes. The factors triggering sexually dimorphic expression of these genes are unknown, and whether the gonadotropin hormones are involved in early gonadal sex differentiation of the Nile tilapia has been unclear. In the present study, we determined the precise timing of expression of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in the pituitary and that of their receptors (fshra and lhcgrbb) in the undifferentiated gonad in both XX and XY tilapia fry by quantitative RT-PCR and immunohistochemical analysis. Expression of fshb mRNA and Fsh protein in the pituitary was detected from the first sampling day (3 dah) to 25 dah in both XX and XY tilapia larvae without sexual dimorphism and increased gradually after 25 dah in the pituitary. fshra mRNA was expressed beginning 5 dah and wa...

4-Guanidinobutyrate amidinohydrolase (EC 3.5.3.7) was purified about 360-fold to apparent homogeneity from Pseudomonas sp. ATCC 14676. The molecular weight of the enzyme was estimated to be 180, 000_??_186, 000 by gel filtration procedure and the method of Hedrick and Smith. The subunit molecular weight was estimated to be 33, 000_??_36, 000 by SDS-polyacrylamide gel electrophoresis. The enzyme was optimally active at pH 10.2 in sodium carbonate buffer. EDTA inactivated the enzyme during incubation at 50°C in phosphate buffer (pH 7.0). Incubation of the inactivated enzyme with Mn2+ restored full activity. The enzyme was inactivated with PCMB, and the PCMB-inactivated enzyme was reactivated by incubation with 2-mercaptoethanol. The enzyme specifically acted toward 4-guanidinobutyrate; 5-guanidinovalerate and 6-guanidinocaproate were hydrolyzed at very low rates. The Km value for 4-guanidinobutyrate was 33mM. Propionate and n-butyrate were competitive inhibitors of the enzyme with Ki values of 2.0mM and 0.5mM, respectively. These properties were compared with those of the analogous guanidinoacetate amidinohydrolase from the same organism.

In the presence of citrate, citrate-fermenting Streptococcus sp. KSM-1106 and KSM-1112 grew in long chains with innumerable cells. Divalent cations, such as Mn2+ and Ca2+, arrested the chain elongation. The results with citrate-negative and citrate-resistant variants suggested the involvement of citrate in the cell separation system. A dechaining activity, which was inhibited by citrate, could be demonstrated in the cell extract of strain KSM-1106. Such a special effect of citrate was not observed on the growth of strains of other lactic streptococci examined.

An organism capable of producing an alkaline proteinase was isolated and identified as Pseudomonas maltophilia.By simple Sephadex-gel chromatographies, the alkaline proteinase was purified to homogeneity with a specific activity 74-fold higher than that of the culture broth. The purified enzyme had a molecular weight of 46,000 consisting of 19,000 and 27,000 molecular subunits. The optimum pH and temperature were pH 10.5 and 55°C, respectively. Calcium ion activated and stabilized the enzyme activity. The enzyme was inhibited by ethylenediaminetetraacetate, phenylmethylsulfonyl fluoride and chymostatin, indicating that the enzyme was a serine metalloenzyme.

The water-extracts of 31 herb species been screened as to suppressive activity toward the mutagenicity of one amino acid pyrolysate, 3-amino-1-methyl-5H-pyrido [4,3-b] indole (Trp-P-2)

A patient with an intracranial embryonal carcinoma, which was resistant to cisplatin-based combined chemotherapy and radiotherapy, developed spinal seeding. Alpha fetoprotein (AFP), human chorionic gonadotropin (HCG) and the beta chain of HCG (NCG beta) in serum or cerebrospinal fluid immediately returned to elevated levels despite sequential chemotherapy and resection. Accordingly, we treated this patient with spinal irradiation and intensive combination chemotherapy (cisplatin etoposide, vinblastine, cyclophosphamide and actinomycin D) followed by autologous bone marrow transplantation. With this treatment, the clinical manifestations showed a tendency to improve and, furthermore, the serum levels of AFP and HCG returned to normal. These findings suggest autologous bone marrow transplantation to be a potential approach to the treatment of patients with embryonal carcinomas.