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Objectives: Eltrombopag, a thrombopoietin-receptor agonist, stimulates hematopoiesis in patients with acquired aplastic anemia (AA). Cytomorphologic changes in bone marrow after eltrombopag administration are still unclear. This study examined the effect of eltrombopag on cytomorphologic findings using data from prior phase 2 studies (E1201 and E1202)., Methods: Microscopic examinations were performed in 31 patients with AA (E1201 [n = 21], E1202 [n = 10]). The relationship between hematologic improvement and morphologic findings was also investigated., Results: In 5 patients (E1201 [n = 3], E1202 [n = 2]), the bone marrow blast count increased after initiation of eltrombopag treatment compared with screening values. The blast count was less than 5%, and the increase in bone marrow blasts was transient in all 4 patients who had bone marrow examinations at follow-up. In 8 patients (E1201 [n = 5], E1202 [n = 3]), dysplastic forms of megakaryocytes were found in the bone marrow following treatment initiation. Dysmegakaryopoiesis of 10% or more was found in 3 patients. None of the patients revealed micromegakaryocytes. Ten patients showed an increase in bone marrow blasts and/or dysmegakaryopoiesis following treatment initiation. Nine of 10 patients showed hematologic improvement in 1 or more lineages., Conclusions: Dysmegakaryopoiesis without micromegakaryocytes and a transient increase of less than 5% in bone marrow blast count may be signs of hematologic improvement with eltrombopag for patients with AA., (© American Society for Clinical Pathology, 2022.)

Many bacteria swim by rotating flagella. The chemotaxis system controls the direction of flagellar rotation. Vibrio alginolyticus, which has a single polar flagellum, swims smoothly by rotating the flagellar motor counterclockwise (CCW) in response to attractants. In response to repellents, the motor frequently switches its rotational direction between CCW and clockwise (CW). We isolated a mutant strain that swims with a CW-locked rotation of the flagellum, which pulls rather than pushes the cell. This CW phenotype arises from a R49P substitution in FliM, which is the component in the C-ring of the motor that binds the chemotaxis signalling protein, phosphorylated CheY. However, this phenotype is independent of CheY, indicating that the mutation produces a CW conformation of the C-ring in the absence of CheY. The crystal structure of FliM with the R49P substitution showed a conformational change in the N-terminal α-helix of the middle domain of FliM (FliMM). This helix should mediates FliM-FliM interaction. The structural models of wild type and mutant C-ring showed that the relatively small conformational change in FliMM induces a drastic rearrangement of the conformation of the FliMM domain that generates a CW conformation of the C-ring., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Bacteria have evolved various types of flagellum, an organella for bacterial motility, to adapt to their habitat environments. The number and the spatial arrangement of the flagellum are precisely controlled to optimize performance of each type of the flagellar system. Vibrio alginolyticus has a single sheathed flagellum at the cell pole for swimming. SflA is a regulator protein to prevent peritrichous formation of the sheathed flagellum, and consists of an N-terminal periplasmic region, a transmembrane helix, and a C-terminal cytoplasmic region. Whereas the cytoplasmic region has been characterized to be essential for inhibition of the peritrichous growth, the role of the N-terminal region is still unclear. We here determined the structure of the N-terminal periplasmic region of SflA (SflAN) at 1.9-Å resolution. The core of SflAN forms a domain-swapped dimer with tetratricopeptide repeat (TPR)/Sel1-like repeat (SLR) motif, which is often found in the domains responsible for protein-protein interaction in various proteins. The structural similarity and the following mutational analysis based on the structure suggest that SflA binds to unknown partner protein by SflAN and the binding signal is important for the precise control of the SflA function., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

The flagellar motor is embedded in the cell envelope and rotates upon interaction between the stator and the rotor. The rotation is powered by ion flow through the stator. A single transmembrane protein named FliL is associated with torque generation in the flagellar motor. We established an Escherichia coli over-expression system for FliL of Vibrio alginolyticus, a marine bacterium that has a sodium-driven polar flagellum. We successfully expressed, purified, and crystallized the ca. 17 kDa full-length FliL protein and generated a construct that expresses only the ca. 14 kDa periplasmic region of FliL (ΔTM FliL). Biochemical characterization and NMR analysis revealed that ΔTM FliL weakly interacted with itself to form an oligomer. We speculate that the observed dynamic interaction may be involved in the role of FliL in flagellar motor function., (© The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Background: Although androgen deprivation therapy for advanced prostate cancer initially exerts excellent anticancer effects, most prostate cancer treated with androgen deprivation therapy eventually recurs as castration-resistant prostate cancer (CRPC). Although aberrant kinase activation has been proposed as a mechanism of castration resistance, comprehensive kinase profiles in CRPC remain unknown. Therefore, we aimed to elucidate the kinome in CRPC as well as the role of key molecules., Methods: We utilized a kinome array in androgen-dependent LNCaP and castration-resistant CxR cells. The effect of Y-box binding protein-1 (YB-1) on androgen receptor (AR) expression was examined by quantitative polymerase chain reaction and western blot analysis. The association between polymorphisms in the YB-1 gene determined by genotyping and YB-1 expression evaluated by immunohistochemistry in prostate cancer tissues, as well as outcome in metastatic prostate cancer, were investigated by the Cochran-Armitage test and the Cox proportional hazards model, respectively. All statistical tests were two-sided., Results: One hundred fifty-six of 180 kinase phosphorylation sites, including ERK and RSK, were activated in CRPC cells, leading to increased phosphorylation of YB-1, which is a key molecule in the progression to CRPC. YB-1 signaling regulated AR V7 expression, and YB-1 inhibition augmented the anticancer effect of enzalutamide. Moreover, polymorphism (rs12030724) in the YB-1 gene affected YB-1 expression in 93 prostate cancer tissues (YB-1 positive rate; 14.3% in TT, 40.0% in AT, and 52.9% in AA, P = .04) and associated with probability of progression in 104 metastatic prostate cancer case patients (AT/TT vs AA, hazard ratio = 0.49, 95% confidence interval = 0.32 to 0.77, P = .001)., Conclusions: YB-1 appears to be a promising target to inhibit the development of castration resistance, even at the AR variant-expressing stage. Polymorphism in the YB-1 gene may be a promising predictive biomarker in hormonal therapy., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

L-Lysine α-oxidase (LysOX) from Trichoderma viride is a homodimeric 112 kDa flavoenzyme that catalyzes the oxidative deamination of L-lysine to form α-keto-ε-aminocaproate. LysOX severely inhibited growth of cancer cells but showed relatively low cytotoxicity for normal cells. We have determined the cDNA nucleotide sequence encoding LysOX from T. viride. The full-length cDNA consists of 2,119 bp and encodes a possible signal peptide (Met1-Arg77) and the mature protein (Ala78-Ile617). The LysOX gene have been cloned and heterologously expressed in Streptomyces lividans TK24 with the enzyme activity up to 9.8 U/ml. The enzymatic properties of the purified recombinant LysOX, such as substrate specificity and thermal stability, are same as those of native LysOX. The crystal structure of LysOX at 1.9 Å resolution revealed that the overall structure is similar to that of snake venom L-amino acid oxidase (LAAO), and the residues involved in the interaction with the amino or carboxy group of the substrate are structurally conserved. However, the entrance and the inner surface structures of the funnel to the active site, as well as the residues involved in the substrate side-chain recognition, are distinct from LAAOs. These structural differences well explain the unique substrate specificity of LysOX., (© The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Unlabelled: There is very little information on the fungistatic or fungicidal effect of visible light. This study investigated the effect of 405-nm light, generated by a light-emitting diode array, on the economically important fungus Botrytis cinerea. The mycelial growth of B. cinerea was inhibited to the greatest extent by light at 405 and 415 nm and was negligibly inactivated at 450 nm, suggesting the presence of a photosensitizing compound that absorbs light mainly at wavelengths of 405-415 nm. Delta-aminolevulinic acid, a precursor of endogenous photosensitizer porphyrins, was used to determine the role of these porphyrins in 405-nm light-mediated photoinactivation of the fungus. Concentration-dependent inhibition of spore germination by delta-aminolevulinic acid and accumulation of singlet oxygen in the spores was observed when the spores were exposed to 405-nm light. These results suggest that the excitation of endogenous porphyrins and subsequent accumulation of singlet oxygen could partially explain the 405-nm light-mediated photoinactivation of B. cinerea. The development of symptoms in detached tomato leaves inoculated with B. cinerea spores was significantly reduced by irradiation with 405-nm light, indicating that 405-nm light has a potential use for controlling plant diseases caused by B. cinerea., Significance and Impact of the Study: Grey mould (Botrytis cinerea) is a very successful necrotroph, causing serious losses in more than 200 crop hosts. This study investigated the antifungal effect of 405-nm light on this pathogen. Our results suggest that the excitation of endogenous porphyrins and subsequent accumulation of singlet oxygen contribute to the 405-nm light-mediated photoinactivation of grey mould. The development of symptoms in detached tomato leaves inoculated with B. cinerea spores was significantly inhibited by irradiation with 405-nm light, indicating that this wavelength of light has a potential use in controlling plant diseases caused by B. cinerea., (© 2014 The Society for Applied Microbiology.)

Objective: To clarify the risk factors and develop a refined risk-stratification model to help in the appropriate selection of docetaxel chemotherapy in patients with castration-resistant prostate cancer., Methods: This study included 97 Japanese patients with castration-resistant prostate cancer who were treated with 70-75 mg/m(2) docetaxel and 10 mg prednisone every 3 or 4 weeks from 2008 to 2013. The oncological outcomes and prognostic significance of clinicopathological factors were analyzed, and significant prognostic factors were used to develop a risk-stratification model., Results: Prostate-specific antigen decline was observed in 75 patients (77.3%), including 43 (44.3%) who achieved a prostate-specific antigen decline of ≥ 50%. The median progression-free survival and overall survival were 5.1 and 20.8 months, respectively. Univariate analysis identified performance status, alkaline phosphatase value, visceral metastasis, duration from diagnosis, duration from initiation of hormone treatment and prior treatment with estramustine as significant predictors of overall survival. Among these, alkaline phosphatase value, visceral metastasis and duration from initiation of hormone treatment were independent prognostic factors in multivariate analysis. Furthermore, risk classification according to the number of independent risk factors present effectively stratified survival among docetaxel-treated castration-resistant prostate cancer patients., Conclusions: Oncologic outcomes in Japanese patients with castration-resistant prostate cancer receiving docetaxel chemotherapy were comparable to or slightly better than those in Western populations, and the risk-stratification model developed in this study may help to predict prognosis and contribute to the selection of suitable therapy after castration resistance., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The proteolytic cascade is the key step in transactivation of sterol regulatory element-binding proteins (SREBPs), a transcriptional factor of lipid synthesis. Proteolysis of SREBP-2 is strictly regulated by sterols, but that of SREBP-1c was not strongly sterol-regulated, but inhibited by polyunsaturated fatty acids (PUFAs). In this study, the proteolytic processing of SREBP-1 and -2 was examined by transfection studies of cDNA-encoding mutants in which all the known cleavage sites were disrupted. In cultured cells, sterol-regulated SREBP-2 processing was completely eliminated by mutation of cleavage sites. In contrast, the corresponding SREBP-1c mutants as well as wild type exhibited large amounts of cleaved products in the nuclear extracts from culture cells and murine liver in vivo. The nuclear form of the mutant SREBP-1c was induced by delipidated condition and suppressed by eicosapentaenoic acid, an n-3 PUFA, but not by sterols. This novel processing mechanism was affected by neither SREBP cleavage-activating protein (SCAP) nor insulin-induced gene (Insig)-1, unlike SREBP-2, but abolished by a serine protease inhibitor. Through analysis of deletion mutant, a site-2 protease recognition sequence (DRSR) was identified to be involved in this novel processing. These findings suggest that SREBP-1c cleavage could be subjected to a novel PUFA-regulated cleavage system in addition to the sterol-regulatory SCAP/Insig system.

The pH dependence and kinetics parameters of renin-angiotensinogen reactions were determined using wild-type and S84G mutant human renins and wild-type and H13Y mutant sheep angiotensinogens. It is explained in this report that (i) renin catalyzes acidic and basic reactions of which the optimum pHs are 5.5 and 7.5-8.2 respectively, both of which produce angiotensin I; (ii) Ser84 specific to human renin accelerates the acidic reaction by 75-110% through elevation of V(max), and shifts the optimum pH of the basic reaction from 7.5 to 8.0-8.2; and (iii) His13 specific to sheep angiotensinogen accelerates the acidic and basic reactions by 25-42% through reduction of K(m). It is concluded from these results that the coexistence of Ser84 in renin and His13 in angiotensinogen brings a pH profile of two separate peaks at pHs 5.5 and 8.2 to the reaction of human renin and sheep angiotensinogen.

An isocitrate dehydrogenase (ICDH) with an unique coenzyme specificity from Acidithiobacillus thiooxidans was purified and characterized, and its gene was cloned. The native enzyme was homodimeric with a subunit of M(r) 45000 and showed a 78-fold preference for NAD(+) over NADP(+). The cloned ICDH gene (icd) was expressed in an icd-deficient strain of Escherichia coli EB106; the activity was found in the cell extract. The gene encodes a 429-amino acid polypeptide and is located between open reading frames encoding a putative aconitase gene (upstream of icd) and a putative succinyl-CoA synthase beta-subunit gene (downstream of icd). A. thiooxidans ICDH showed high sequence similarity to bacterial NADP(+)-dependent ICDH rather than eukaryotic NAD(+)-dependent ICDH, but the NAD(+)-preference of the enzyme was suggested due to residues conserved in the coenzyme binding site of the NAD(+)-dependent decarboxylating dehydrogenase.

The phenotypes of infiltrating lymphocytes in liver with chronic hepatitis C, including changes associated with interferon (IFN) treatment, were characterized. Specimens obtained from 22 patients treated with IFN were examined using avidin-biotin-peroxidase immunohistochemistry. In areas of lobular and periportal inflammation, most lymphocytes were CD8+ T cells of the CD45RO+ (memory) subset. The centres of lymphoid follicles were occupied by CD20+ B cells and a few CD4+ T cells which were CD45RA+ (naive subset). Follicular centres were surrounded mainly with CD4+ T cells. CD8+ T cells, mostly CD45RO+, were scattered through the mantle zones of follicles and extended around them. No significant changes in CD45RA+ lobular infiltrates accompanied IFN treatment. On the other hand, the number of CD45RO+ lobular infiltrates decreased after IFN treatment in complete responders (P < 0.01). Moreover, there were significant correlations between CD45RO+ cell counts and serum alanine aminotransferase concentrations, CD45RO+ cell counts and the liver histologic grade and CD45RO+ cell counts and CD8+ cell counts. These results suggest that CD8+ memory T cells participate in hepatocyte injury in chronic hepatitis C, and that a decrease of CD8+ memory T cells correlates with the decreased liver inflammation with IFN treatment.

Since the degradation of uterine cervical extracellular matrix is an essential process in cervical ripening and dilatation at term, we examined the effect of pregnancy on the level of pro-matrix metalloproteinase 9 (proMMP-9)/progelatinase B in the rabbit uterine cervix and its regulation in uterine cervical fibroblasts. Uterine cervices shortly before term parturition contained high levels of proMMP-9 compared with those of nonpregnant rabbits. Uterine cervical fibroblasts prepared from rabbits at 23 days of gestation did not produce proMMP-9; in contrast, interleukin (IL)-1 alpha, IL-1 beta, or phorbol 12-myristate 13-acetate (PMA) induced proMMP-9 production along with an increase of proMMP-9 mRNA, and the IL-1-mediated induction of proMMP-9 was synergistically enhanced by PMA. On the other hand, physiological concentrations of progesterone suppressed IL-1-and/or PMA-mediated production of proMMP-9 in a dose-dependent manner. This suppressive effect of progesterone on proMMP-9 production was accompanied by a decrease in matrix metalloproteinase 9 (MMP-9) mRNA, indicating that progesterone down-regulates the production of proMMP-9 at the transcriptional level. These results indicate that in the rabbit uterine cervix, IL-1 alpha and IL-1 beta are effective inducers of the proMMP-9 production, and progesterone is a physiological suppressor. Thus, MMP-9 and progesterone are very likely to play essential roles in cervical ripening and dilatation in the rabbit, and the production of MMP-9 in the cervix is finely regulated during pregnancy.

Clones of a gene encoding an endo-1,4-beta-glucanase (EC 3.2.1.4) were obtained from Bacillus sp. 22-28, and the nucleotides were sequenced. A recombinant plasmid, pMK5, included a complete ORF of 2352 bp that encoded 783 amino acid residues. Another plasmid, pM3, which showed enzymatic activity in E. coli JM109, was also obtained, and it included an incomplete ORF of 1873 bp, which lacked the original stop codon and 479 bp of the C-terminal region. The enzymes purified from both of the two types of transformants have shown almost the same properties in comparison with that of the wild type Bacillus sp. 22-28.

The chain conformation of an arabinoxylan isolated from the rice endosperm cell wall, which was composed of the linear backbone of a 1,4-linked beta-xylose residue highly substituted at the C3 position with alpha-L-arabinofuranose, was studied by X-ray fiber diffraction and a conformational analysis. From the X-ray results the polysaccharide was suggested to take an extended left-handed, three-fold helical structure which is not a desirable conformation to make a complex firmly associated with cellulose or xyloglucan that was elucidated to be present in the cell wall. A conformational analysis, using the PFOS force field for the O-alpha-L-arabinofuranose-(1-->3)-beta-D-xylose residue and O-alpha-L-arabinofuranose-(1-->3)-[(1-->4)-beta-D-xylose residue]3, in which the arabinose residue was linked to the C3 position of the middle residue of the xylotriose residue, and using the PS79 computer program for the arabinoxylan indicated that the backbone xylan chain could not take a two-fold helix similar to that of cellulose because of significant steric interaction between the arabinose side chain and the backbone. Thus, the left-handed, three-fold helix was found to be reasonable.

The effects of chemicals such as metal ions and typical organic enzyme inhibitors on the activity of deglycosylated β-fructofuranosidases (P-1 and P-2) from Aureobasidium were observed and compared with those of native enzymes. P-1 and P-2 enzymes became sensitive to metal ions, such as Fe 2+ , Co 2+ , Ni 2+ and Al 3+ , after deglycosylation. The enzymes were also inhibited by a sulphydryl reagent (monoiodoacetic acid), a peptide-hydrolysing reagent (hydroxylamine) and chelating reagents (sodium citrate, EDTA and sodium azide) after deglycosylation. The importance of deglycosylation for the determination of the true characteristics of the enzymes against chemicals is discussed.

A new type of chimeric 3-isopropylmalate dehydrogenase (2T2M6T) was produced by expressing the fused gene of Bacillus subtilis and Thermus thermophilus. The enzyme shows heat stability intermediate between those of the parents. The crystal of the enzyme belongs to the space group of P3(2)21, with cell dimensions of a = b = 78.9 A and c = 158.9 A. Two thermostable mutants of the chimeric enzyme were prepared by site-directed mutagenesis and then crystallized.

Guanidinobutyrase (guanidinobutyrate amidinohydrolase, EC. 3.5.3.7) catalyzing the third step of the arginine oxygenase pathway in Brevibacterium helvolum IFO 12073 (ATCC 11822) was purified to homogeneity and characterized. The enzyme had a molecular weight of 190,000 and was composed of four apparently identical subunits with a molecular weight of 45,000. The E(1%) value at 280 nm of the enzyme protein was 2.4. The enzyme contained 0.5 mol of firmly bound Zn(2+) per mol of subunit. The enzyme was highly specific for 4-guanidinobutyrate, but had a weak activity toward L- and D-arginine. The Michaelis constant (Km) for 4-guanidinobutyrate was 2.9 mM. The optimum pH was 9.0. Strong mixed type inhibition was observed with thioglycolate and several other thiol compounds. These properties were compared with those of the enzyme of fluorescent Pseudomonas and discussed.

A chimeric gene was constructed by fusing the Bacillus subtilis and Thermus thermophilus genes coding for 3-isopropylmalate dehydrogenase, and expressed in Escherichia coli. The chimeric enzyme was crystallized in a size suitable for X-ray structure analysis. The crystal has a space group of P3(1)21 or P3(2)21, a = b = 77.1 A and c = 158.3 A, which is isomorphous with that of the native enzyme from T. thermophilus.