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The imbalance in the phosphorylation and the dephosphorylation of proteins leads to various diseases. Therefore, in vivo, the functions of protein kinases and protein phosphatases are strictly regulated. Mg 2+ /Mn 2+ -dependent protein phosphatase PPM1M has been implicated in immunity and cancer; however, the regulation mechanism remains unknown. In this study, we show that PPM1M is regulated in different ways by multiple phosphorylation. PPM1M has four Ser/Thr-Pro motifs (Ser27, Ser43, Ser60, and Thr254) that are recognized by proline-directed kinases, and Ser60 was found to be phosphorylated by cyclin-dependent kinase 5 (CDK5) in the cell. The phospho-mimetic mutation of Ser27 and Ser43 in the N-terminal domain suppresses the nuclear localization of PPM1M and promotes its accumulation in the cytoplasm. The phospho-mimetic mutation of Ser60 decreases PPM1M activity; conversely, the phospho-mimetic mutation of Thr254 increases PPM1M activity. These results suggest that the subcellular localization and phosphatase activity of PPM1M are regulated by protein kinases, including CDK5, via phosphorylation at multiple sites. Thus, PPM1M is differentially regulated by proline-directed kinases, including CDK5., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Bone morphogenetic protein 2 (BMP2)-inducible kinase (BMP2K) is induced by the cytokine BMP2, which is also implicated in the production of bone differentiation. In addition to regulating bone differentiation, BMP2K is implicated in a variety of cancers. Therefore, understanding the variables that determine where in the cell this kinase functions may help in understanding malignancies linked to BMP2K. However, the mechanisms regulating the subcellular localization of BMP2K are mainly unknown. By liquid-liquid phase separation (LLPS), BMP2K forms droplets in the cytoplasm, but how the droplets are regulated remains unclear. The reason why BMP2K localizes to the cytoplasm irrespective of having a nuclear localization signal (NLS) is also unknown. Here we show the element that controls BMP2K's LLPS and cytoplasmic localization. A glutamine-rich area is necessary for BMP2K phase separation, and droplet formation is controlled by hyperosmolarity. Cytoplasmic localization of BMP2K is managed by inhibition of NLS function through phosphorylation of Ser-1010 and by a newly found cytoplasmic localization region that antagonizes the NLS. These results will provide an important biochemical foundation for the advancement of BMP2K-related cell biology, structural biology, and pathophysiology., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

CaMK phosphatase (CaMKP/POPX2/PPM1F) is a Ser/Thr protein phosphatase that belongs to the PPM family. Accumulating evidence suggests that CaMKP is involved in the pathogenesis of various diseases, including cancer. To clarify the relationship between CaMKP activity and human breast cancer cell motility, we examined the phosphatase activity of CaMKP in cell extracts. CaMKP activity assays of the immunoprecipitates prepared from the cell extract revealed that cells exhibiting higher motility had higher CaMKP activity, with no significant differences in the specific activity being observed. Two CaMKP-specific inhibitors, 1-amino-8-naphthol-4-sulfonic acid (ANS) and 1-amino-8-naphthol-2,4-disulfonic acid (ANDS), inhibited the migration of highly invasive MDA-MB-231 breast cancer cells without significant cytotoxicity, while an inactive analog, naphthionic acid, did not. Furthermore, the cells lost their elongated morphology and assumed a rounded shape following treatment with ANS, whereas they retained their elongated morphology following treatment with naphthionic acid. Consistent with these findings, ANS and ANDS significantly enhanced the phosphorylation level of CaMKI, a cellular substrate of CaMKP, while naphthionic acid did not. The present data suggest that CaMKP could be a novel therapeutic target for cancer metastasis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

CaMK phosphatase (CaMKP/PPM1F/POPX2) is a Mn 2+ -dependent, calyculin A/okadaic acid-insensitive Ser/Thr protein phosphatase that belongs to the PPM family. CaMKP is thought to be involved in regulation of not only various protein kinases, such as CaM kinases and p21-activated protein kinase, but also of cellular proteins regulated by phosphorylation. A large-scale screening of a chemical library identified gallic acid and some of its alkyl esters as novel CaMKP inhibitors highly specific to CaMKP. Surprisingly, they caused specific carbonylation of CaMKP, leading to its inactivation. Under the same conditions, no carbonylation nor inactivation was observed when PPM1A, which is affiliated with the same family as CaMKP, and λ-phosphatase were used. The carbonylation reaction was inhibited by SH compounds such as cysteamine in a dose-dependent manner with a concomitant decrease in CaMKP inhibition by ethyl gallate. The pyrogallol structure of gallate was necessary for the gallate-mediated carbonylation of CaMKP. Point mutations of CaMKP leading to impairment of phosphatase activity did not significantly affect the gallate-mediated carbonylation. Ethyl gallate resulted in almost complete inhibition of CaMKP under the conditions where the carbonylation level was nearly identical to that of CaMKP carbonylation via metal-catalyzed oxidation with ascorbic acid/FeSO 4 , which resulted in only a partial inhibition of CaMKP. The gallate-mediated carbonylation of CaMKP absolutely required divalent cations such as Mn 2+ , Cu 2+ , Co 2+ and Fe 2+ , and was markedly enhanced by a phosphopeptide substrate. When MDA-MB-231 cells transiently expressing CaM kinase I, a CaMKP substrate, were treated by ethyl gallate, significant enhancement of phosphorylation of CaM kinase I was observed, suggesting that ethyl gallate can penetrate into cells to inactivate cellular CaMKP. All the presented data strongly support the hypothesis that CaMKP undergoes carbonylation of its specific amino acid residues by incubation with alkyl gallates and the divalent metal cations, leading to inactivation specific to CaMKP., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Protein phosphatase PPM1H is known to participate in various biological or pathophysiological mechanisms. However, little is known about the molecular mechanisms of its regulation. In this study, we investigated the protein kinases that directly phosphorylate PPM1H, identifying them as cAMP-dependent protein kinase (PKA) and Ca 2+ /calmodulin-dependent protein kinase I (CaMKI). In vitro and in silico analyses showed that the phosphorylation sites of PPM1H by PKA and CaMKI were Ser-123 and Ser-210, respectively. The phosphorylation state of PPM1H in cells exhibited the kinase activator- and inhibitor-dependent changes. In mouse neuroblastoma Neuro2a cells, phosphorylation of Ser-210 was much higher in the phospho-mimetic mutant (S123D) than in the non-phosphorylatable mutant (S123A) when they were treated with ionomycin. This suggests that a hierarchical phosphorylation, with initial phosphorylation of Ser-123 promoting subsequent phosphorylation of Ser-210, occurs in these neuron-like cells. Moreover, in cell-based assay a PPM1H(S123A/S210A) double mutant barely dephosphorylated Smad1, a transcription factor known as an endogenous substrate of PPM1H. These results suggest that cAMP and Ca 2+ /calmodulin regulate dephosphorylation of Smad1 through the dual phosphorylation of PPM1H at Ser-123 and Ser-210., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Ca 2+ /calmodulin-dependent protein kinase I isoforms (CaMKIα, β, γ, and δ) play important roles in Ca 2+ signaling in eukaryotic cells by being activated by CaMK kinase (CaMKK) through phosphorylation at a Thr residue in the activation loop. However, we have recently found that, unlike rat CaMKIα (rCaMKIα), C-terminally truncated fragments of zebrafish and mouse CaMKIδ [zCaMKIδ(1-299) and mCaMKIδ(1-297)] produced by Escherichia coli exhibit almost full activity in the absence of CaMKK. To address the CaMKK-independent activation mechanism of CaMKIδ in E. coli cells, here we performed comparative analyses between recombinant zCaMKIδ(1-299) and rCaMKIα(1-294) in vitro. By using a kinase-dead mutant of zCaMKIδ(1-299) and λ phosphatase coexpression method, we elucidated that zCaMKIδ(1-299) was highly autophosphorylated and activated in E. coli during cell culture, but rCaMKIα(1-294) was not. The major autophosphorylation site leading to activation of the kinase was Ser 296 , determined using mass spectrometry analysis in conjunction with site-directed mutagenesis. Furthermore, mimicking phosphorylation at Ser 296 in full-length zCaMKIδ resulted in additional activation of the kinase compared with CaMKI fully activated by CaMKK. Our results provide the first evidence that CaMKIδ is activated through CaMKK-independent phosphorylation at Ser 296 , which might be a clue to understand the physiological regulation of CaMKIδ isoform., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Casein kinase 1 (CK1) is a widely expressed Ser/Thr kinase in eukaryotic organisms that is involved in various cellular processes (e.g., circadian rhythm and apoptosis). Therefore, preparing highly active CK1 and investigating its properties in vitro have important implications for understanding the biological roles of the kinase. However, recombinant CK1 undergoes autoinactivation via autophosphorylation in Escherichia coli cells and thus is undesirably prepared as a phosphorylated and inactivated kinase. To circumvent this problem, we established a protein expression system using E. coli strain BL21(DE3)pλPP in which λ protein phosphatase (λPPase) is constitutively expressed. Using this system, recombinant CK1 isoforms (α, δ and ε) were readily prepared as unphosphorylated forms. Furthermore, we found that CK1s prepared using BL21(DE3)pλPP showed markedly higher activity than those prepared by the conventional BL21(DE3). Finally, we demonstrated that the kinase activity of CK1δ from BL21(DE3)pλPP was higher than that prepared by a conventional method consisting of troublesome steps such as in vitro λPPase treatment. Thus, this simple method using BL21(DE3)pλPP is valuable for preparing highly active CK1s. It may also be applicable to other kinases that are difficult to prepare because of phosphorylation in E. coli cells., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Carbapenemase-producing Enterobacteriaceae (CPE) are increasing worldwide. Rapid and accurate detection of CPE is necessary for appropriate antimicrobial treatment and hospital infection control. However, CPE contains some strains that are difficult to detect depending on genotype and MIC value of carbapenem, and a detection method has not been established. The recently reported modified carbapenem inactivation method (mCIM) has been developed in CLSI M100-S27 as a phenotypic technique for detecting carbapenemase activity. In the present study, we examined mCIM as a new CPE detection method using 207 Enterobacteriaceae isolates in comparison with the three existing screening methods of modified Hodge test, Carba NP test and carbapenem inactivation method and evaluated its performance. Consequently, both the sensitivity and specificity of mCIM were 100%, indicating better results than the conventional screening methods. The mCIM is a useful tool for microbiology laboratories due to its simplicity, clear criteria, cost-effectiveness and availability at any laboratory., (Copyright © 2017 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

The 7-valent pneumococcal conjugate vaccine (PCV7) was introduced among children in Japan in 2010. There are no long-term multicenter surveillance studies of antimicrobial resistance in S. pneumoniae before and after the introduction of PCV7. Therefore, we examined chronological trends in antimicrobial resistance among 4534 strains of S. pneumoniae isolated from both children and adults in the Kinki region of Japan during 2001-2015. High-level penicillin and third-generation cephalosporin resistance in S. pneumoniae increased among both children and adults during the period before the introduction of PCV7 (2001-2010). Besides penicillin and cephalosporin, pneumococcal carbapenem and macrolide resistance increased among children. The rate of resistance to these antibiotics was higher among children than among adults. The introduction of PCV7 decreased the rate of non-susceptibility to β-lactam antibiotics and the rate of multidrug resistant S. pneumoniae among children, but not among adults., (Copyright © 2018 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Intracellular signal transduction is built on the basis of the subtle balance between phosphorylation and dephosphorylation. Ca 2+ /calmodulin-dependent protein kinase phosphatase (CaMKP/PPM1F/POPX2) and CaMKP-N (PPM1E/POPX1) are Ser/Thr phosphatases that belong to the PPM (protein phosphatase, Mg 2+ /Mn 2+ -dependent) family. The former was discovered in rat brain as a novel protein phosphatase regulating Ca 2+ /calmodulin-dependent protein kinases (CaMKs), whereas the latter was first identified in human cDNA databases using the rat CaMKP sequence. Subsequent studies have revealed that they are involved in various cellular functions through regulation of not only CaMKs but also other protein kinases such as AMP-activated protein kinase. Furthermore, accumulating evidence shows possible involvement of CaMKP and CaMKP-N in the pathogenesis of various diseases including cancer. Therefore, the biochemistry of CaMKP and CaMKP-N largely contributes to molecular medicine targeting these phosphatases. In this review, we summarized recent progress in the enzymology and biology of CaMKP and CaMKP-N. We also focused on etiology studies in which CaMKP and CaMKP-N are involved. Based on the emerging evidence, future perspectives of studies on these phosphatases and related issues to be elucidated are discussed., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Cyclin-dependent kinase-like 5 (CDKL5) is a Ser/Thr protein kinase primarily expressed in the central nervous system and is known to cause X-linked neurodevelopmental disorders such as Rett syndrome. However, the mechanisms regulating CDKL5 have not yet been fully clarified. Therefore, in this study, we investigated the protein kinase that directly phosphorylates CDKL5, identifying it as dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A), an enzyme binding to and phosphorylating CDKL5. We showed that subcellular distribution of CDKL5 was regulated by its phosphorylation by DYRK1A. In mouse neuroblastoma Neuro2a cells, CDKL5 was localized in both the cytosol and nucleus, whereas DYRK1A showed a typical nuclear localization. When CDKL5 and DYRK1A were co-expressed, the cytosolic localization of CDKL5 was significantly increased. Results of site-directed mutagenesis revealed that the phosphorylation site was Ser-308, in the vicinity of the nuclear localization signal. A mutation mimicking the phosphorylated serine residue by aspartate substitution (S308D) changed CDKL5 localization to the cytosol, whereas the corresponding alanine-substituted analog, CDKL5(S308A), was primarily localized to the nucleus. Taken together, these results strongly suggested that DYRK1A bound to CDKL5 and phosphorylated it on Ser-308, thus interfering with its nuclear localization., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Six Klebsiella pneumoniae clinical isolates resistant to various cephalosporins and cephamycins were identified in a Japanese general hospital, a tertiary care hospital, between November 2009 and April 2010. All K. pneumoniae isolates carried bla GES-4 and bla SHV-1 , while 2 K. pneumoniae isolates also harbored bla CTX-M-15 . The pulsed-field gel electrophoresis patterns revealed that these 6 K. pneumoniae isolates were almost identical, suggesting their clonal relatedness. Plasmid profiles and conjugation assays revealed that these bla GES-4 genes were located on similar conjugative plasmids. These data indicate that nosocomial spread caused by K. pneumoniae isolates producing bla GES-4 carbapenemase occurred at a Japanese general hospital. K. pneumoniae isolate harboring bla GES-4 is rarely reported in Japan, and, to the best of our knowledge, this is the second report of K. pneumoniae isolates harboring bla GES-4 that occurred nosocomial spread in Japan., (Copyright © 2016. Published by Elsevier Ltd.)

To analyze a variety of protein phosphatases, we developed phosphorylated TandeMBP (P-TandeMBP), in which two different mouse myelin basic protein isoforms were fused in tandem, as a protein phosphatase substrate. P-TandeMBP was prepared efficiently in four steps: (1) phosphorylation of TandeMBP by a protein kinase mixture (Ca(2+)/calmodulin-dependent protein kinase Iδ, casein kinase 1δ, and extracellular signal-regulated kinase 2); (2) precipitation of both P-TandeMBP and protein kinases to remove ATP, Pi, and ADP; (3) acid extraction of P-TandeMBP with HCl to remove protein kinases; and (4) neutralization of the solution that contains P-TandeMBP with Tris. In combination with the malachite green assay, P-TandeMBP can be used to detect protein phosphatase activity without using radioactive materials. Moreover, P-TandeMBP served as an efficient substrate for PPM family phosphatases (PPM1A, PPM1B, PPM1D, PPM1F, PPM1G, PPM1H, PPM1K, and PPM1M) and PPP family phosphatase PP5. Various phosphatase activities were also detected with high sensitivity in gel filtration fractions from mouse brain using P-TandeMBP. These results indicate that P-TandeMBP might be a powerful tool for the detection of protein phosphatase activities., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP/PPM1F) is a Ser/Thr phosphatase that belongs to the PPM family. Growing evidence suggests that PPM phosphatases including CaMKP act as a complex with other proteins to regulate cellular functions. In this study, using the two-dimensional far-western blotting technique with digoxigenin-labeled CaMKP as a probe, in conjunction with peptide mass fingerprinting analysis, we identified neurofilament L (NFL) as a CaMKP-binding protein in a Triton-insoluble fraction of rat brain. We confirmed binding of fluorescein-labeled CaMKP (F-CaMKP) to NFL in solution by fluorescence polarization. The analysis showed that the dissociation constant of F-CaMKP for NFL is 73 ± 17 nM (n = 3). Co-immunoprecipitation assay using a cytosolic fraction of NGF-differentiated PC12 cells showed that endogenous CaMKP and NFL form a complex in cells. Furthermore, the effect of CaMKP on self-assembly of NFL was examined. Electron microscopy revealed that CaMKP markedly prevented NFL from forming large filamentous aggregates, suggesting that CaMKP-binding to NFL inhibits its filament association. These findings may provide new insights into a novel mechanism for regulating network formation of neurofilaments during neuronal differentiation., (Copyright © 2016 Elsevier Inc. All rights reserved.)

We describe here the expression and characterization of a constitutively active fragment of zebrafish Ca(2+)/calmodulin-dependent protein kinase (CaMK) Iδ designated zCaMKIδ(1-299) that lacks an autoinhibitory domain. We used a simple one-step purification method to isolate the recombinant enzyme at high yield (220 mg/l of the culture medium) from the soluble fraction of lysates prepared from Escherichia coli. Unlike the corresponding fragment of CaMKIα (CaMKΙα(1-294)), the kinase activity of zCaMKIδ(1-299), without activation procedures, was comparable to that of wild-type zCaMKIδ activated by CaMK kinase. zCaMKIδ(1-299) exhibited broad substrate specificity highly similar to that of wild-type zCaMKIδ, and complementary to that of the cAMP-dependent protein kinase catalytic subunit (PKAc). The protein kinase activity of zCaMKIδ(1-299) was higher compared with that of PKAc as well as CX-30K-CaMKII that comprises a constitutively active fragment of CaMKII fused to the N-terminal region of Xenopus CaMKI. Furthermore, kinase activity was highly stable against thermal inactivation and repeated freezing-thawing. Thus, zCaMKIδ(1-299) represents a readily available alternative that can be used as a "High-performance phosphorylating reagent" alone or in combination with PKAc in diverse experiments on protein phosphorylation and dephosphorylation., (Copyright © 2016 Elsevier Inc. All rights reserved.)

We surveyed the status of community-acquired infections involving four extended-spectrum β-lactamase (ESBL)-producing bacteria (Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Proteus mirabilis) isolated from clinical specimens from 11 social insurance hospitals in Japan in 2012. These are member hospitals of the Japan Community Healthcare Organization, an independent administrative hospital organization. The isolation rates for E. coli, K. pneumoniae, K. oxytoca, and P. mirabilis were 14.0% (165/1176), 3.3% (16/480), 3.1% (4/130), and 15.9% (17/107), respectively. The CTX-M-9 group, the most frequently detected genotype, was found in 77.0% (127/165) of E. coli and 43.8% (7/16) of K. pneumoniae isolates. Among K. oxytoca isolates, 75% (3/4) were the CTX-M-1 group, and all 17 P. mirabilis strains were the CTX-M-2 group. ESBL-producing bacteria isolation rates in each hospital ranged from 5.8% to 21.5% (median 9.5%), and the proportion of community-acquired infections among ESBL-producing bacteria isolates ranged from 1.6% to 30.8% (median 11.4%) in each hospital. Overall, the rates of ESBL-producing bacterial infection in all community-acquired infections and in all hospital infections were 10.6% (115/1081) and 10.7% (87/812), respectively. The ESBL-producing bacteria are not limited to certain regions or hospitals but are spreading in communities throughout Japan., (Copyright © 2016 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP/PPM1F) is a Ser/Thr protein phosphatase that belongs to the PPM family. It is important to identify an endogenous regulator of CaMKP. Using an Escherichia coli two-hybrid screening method, we identified the C-terminal cytoplasmic fragment of protocadherin γ subfamily C5 (Pcdh-γC5), which was generated by intracellular processing, as a CaMKP-binding protein. Dephosphorylation of phosphorylated Ca(2+)/calmodulin-dependent protein kinase I (CaMKI) by CaMKP was significantly activated by the C-terminal cytoplasmic fragment, Pcdh-γC5(715-944), both in vitro and in cells, suggesting that the C-terminal fragment functions as an endogenous activator of CaMKP. The nuclear translocation of the fragment was blocked by its binding to cytoplasmic CaMKP to form a ternary complex with CaMKI. Taken together, these results strongly suggest that the C-terminal cytoplasmic fragment of Pcdh-γC5 acts as a scaffold for CaMKP and CaMKI to regulate CaMKP activity. These findings may provide new insights into the reversible regulation of CaMKP in cells., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Doublecortin-like protein kinase (DCLK) is a microtubule-associated protein kinase predominantly expressed in brain. In a previous paper, we reported that zebrafish DCLK2 (zDCLK) was cleaved into two functional fragments; the N-terminal zDCLK(DC+SP) with microtubule-binding activity and the C-terminal zDCLK(kinase) with a Ser/Thr protein kinase activity. In this study, we demonstrated that zDCLK(kinase) was widely distributed in the cytoplasm and translocated into the nucleus when the cells were treated under hyperosmotic conditions with NaCl or mannitol. By two-hybrid screening using the C-terminal domain of DCLK, Jun dimerization protein 2 (JDP2), a nuclear transcription factor, was identified as zDCLK(kinase)-binding protein. Furthermore, JDP2 served as an efficient substrate for zDCLK(kinase) only when histone was present. These results suggest that the kinase fragment of DCLK is translocated into the nucleus upon hyperosmotic stresses and that the kinase efficiently phosphorylates JDP2, a possible target in the nucleus, with the aid of histones., (Copyright © 2014 Elsevier Inc. All rights reserved.)

With the increase in extended spectrum β-lactamase (ESBL)-producing bacteria in the community, cases are often seen in which treatment of infectious diseases with oral antimicrobial agents is difficult. Therefore, we measured the antimicrobial activities of 14 currently available oral antimicrobial agents against ESBL-producing Escherichia coli and Klebsiella pneumoniae. Based on the standard of the Clinical and Laboratory Standards Institute (CLSI), E. coli showed high susceptibility rates of 99.4% to faropenem (FRPM). In terms of fluoroquinolones, the susceptibility rate of E. coli to levofloxacin (LVFX) was low at 32.2%, whereas it showed a good susceptibility rate of 93.1% to sitafloxacin (STFX). With respect to other antimicrobial agents, susceptibility rates to fosfomycin (FOM) and colistin (CL) were more than 90% each, whereas rates of the two antimicrobial agents expected as therapeutic agents, minocycline (MINO) and sulfamethoxazole-trimethoprim (ST), were low at 62.4% and 44.3%, respectively. Based on the CLSI standard, K. pneumoniae showed high susceptibility rates to ceftibuten (CETB) (91.89%), LVFX (86.49%), and STFX (94.6%), indicating that K. pneumoniae showed higher rates than those of E. coli, particularly to fluoroquinolones. Comparison of susceptibility rates according to E. coli genotype showed that many antimicrobial agents existed to which the CTX-M-9 group showed high susceptibility rates. However, there were many agents to which the CTX-M-1 group showed low susceptibility rates, particularly to CETB (51.1%) and LVFX (17.0%). Although there was no significant difference by genotype between FRPM, STFX, and FOM, a significant difference was observed between LVFX, MINO, and ST. Antibiotic-resistant bacteria with highly pathogenic strains have spread in the community, appropriate use of oral antimicrobial agents is required., (Copyright © 2013 Japanese Society of Chemotherapy and the Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Ca(2+)/calmodulin-dependent protein kinase Iδ (CaMKIδ) is expressed ubiquitously, but little is known about its physiological functions. Recently, we cloned and characterized two splice variants of zebrafish (Danio rerio) CaMKIδ (CaMKIδ-S/L). In the present study we cloned a new CaMKIδ isoform, CaMKIδ-LL, encoded by a different gene from CaMKIδ-S/L. While the catalytic domain of CaMKIδ-LL showed 86% identity that of CaMKIδ-S/L, it had a unique C-terminal sequence. To clarify the functional role of CaMKIδ-LL, we investigated the biological significance of this new isoform during zebrafish embryogenesis. Although CaMKIδ-LL exhibited essentially the same catalytic properties and substrate specificities as the other CaMKIδ isoforms, it showed different temporal and spatial expression. During zebrafish embryogenesis, RT-PCR analysis detected CaMKIδ-LL expression after 48 h post-fertilization. Western blotting in adult zebrafish demonstrated that CaMKIδ-LL is expressed in the brain, the eye, and, abundantly, in fins. Knockdown of CaMKIδ-LL expression using morpholino-based antisense oligonucleotides resulted in an increase in abnormal embryos with small fins and underdeveloped cartilage. These phenotypes were rescued by co-injection with recombinant CaMKIδ-LL. These results clearly indicated that CaMKIδ-LL plays an important role in the generation of cartilage and fins during zebrafish embryogenesis., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Blue-native electrophoresis (BNE) is a useful technique for analyzing protein complexes, but the Coomassie brilliant blue (CBB) dye used in BNE often hampers in-gel detection of enzymatic activity. Here we report an improved method, termed ink-native electrophoresis (INE), in which Pelikan 4001 fountain pen ink is used as a charge-shifting agent instead of CBB. INE is more suitable than BNE for in-gel detection of protein kinase activity after polyacrylamide gel electrophoresis (PAGE), and its performance in protein complex separation is comparable to that of conventional BNE. INE may provide a powerful tool to isolate and analyze various protein complexes., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Cyclin-dependent kinase-like 5 (CDKL5) is a Ser/Thr protein kinase predominantly expressed in brain and mutations of its gene are known to be associated with neurodevelopmental disorders such as X-linked West syndrome and Rett syndrome. However, the physiological substrates of CDKL5 that are directly linked to these neurodevelopmental disorders are currently unknown. In this study, we explored endogenous substrates for CDKL5 in mouse brain extracts fractionated by a liquid-phase isoelectric focusing. In conjunction with CDKL5 phosphorylation assay, this approach detected a protein band with an apparent molecular mass of 120kDa that is remarkably phosphorylated by CDKL5. This 120-kDa protein was identified as amphiphysin 1 (Amph1) by LC-MS/MS analysis, and the site of phosphorylation by CDKL5 was determined to be Ser-293. The phosphorylation mimic mutants, Amph1(S293E) and Amph1(S293D), showed significantly reduced affinity for endophilin, a protein involved in synaptic vesicle endocytosis. Introduction of point mutations in the catalytic domain of CDKL5, which are disease-causing missense mutations found in Rett patients, resulted in the impairment of kinase activity toward Amph1. These results suggest that Amph1 is the cytoplasmic substrate for CDKL5 and that its phosphorylation may play crucial roles in the neuronal development., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP/PPM1F) is a Ser/Thr protein phosphatase that dephosphorylates and regulates multifunctional Ca(2+)/calmodulin-dependent protein kinases. Although CaMKP is known to be activated by phosphorylation with CaMKII and stimulated by the addition of polycations such as poly-l-lysine, detailed mechanisms of regulation of CaMKP in vivo still remain unclear. In the present study, we found that CaMKP is regulated by oxidation/reduction at Cys residue(s). When CaMKP was incubated with H(2)O(2), time- and dose-dependent inactivation of the enzyme was observed. This inactivation was restored when the inactivated CaMKP was treated with a reducing agent such as 2-mercaptoethanol. Since there are three Cys residues (Cys-259, Cys-315, and Cys-359) in human CaMKP (hCaMKP), we produced three point mutants of hCaMKP, CaMKP(C259S), CaMKP(C315S), and CaMKP(C359S), of which the Cys residues were replaced by Ser residues. Among these Cys-substituted mutants, only CaMKP(C359S) exhibited significant tolerance against oxidation by H(2)O(2). Incubation of CaMKP with H(2)O(2) led to formation of disulfide bond between Cys-359 and Cys-259/Cys-315, resulting in the inactivation of the enzyme. These results suggest that hCaMKP activity is reversibly regulated by oxidation/reduction at Cys-359., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Nuclear Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP-N/PPM1E) is an enzyme that dephosphorylates and downregulates multifunctional Ca(2+)/calmodulin-dependent protein kinases (CaMKs) as well as AMP-dependent protein kinase. In our previous study, we found that zebrafish CaMKP-N (zCaMKP-N) underwent proteolytic processing and translocated to cytosol in a proteasome inhibitor-sensitive manner. In the present study, we found that zCaMKP-N is regulated by phosphorylation at Ser-480. When zCaMKP-N was incubated with the activated CaMKI, time-dependent phosphorylation of the enzyme was observed. This phosphorylation was significantly reduced when Ser-480 was replaced by Ala, suggesting that CaMKI phosphorylates Ser-480 of zCaMKP-N. Phosphorylation-mimic mutants, S480D and S480E, showed higher phosphatase activities than those of wild type and S480A mutant in solution-based phosphatase assay using various substrates. Furthermore, autophosphorylation of CaMKII after ionomycin treatment was more severely attenuated in Neuro2a cells when CaMKII was cotransfected with the phosphorylation-mimic mutant of zCaMKP-N than with the wild-type or non-phosphorylatable zCaMKP-N. These results strongly suggest that phosphorylation of zCaMKP-N at Ser-480 by CaMKI activates CaMKP-N catalytic activity and thereby downregulates multifunctional CaMKs in the cytosol., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP) and its nuclear homolog CaMKP-N are Ser/Thr protein phosphatases that belong to the PPM family. These phosphatases are highly specific for multifunctional CaM kinases and negatively regulate their activities. CaMKP-N is only expressed in the brain and specifically localized in the nucleus. In this study, we found that zebrafish CaMKP-N (zCaMKP-N) underwent proteolytic processing in both the zebrafish brain and Neuro2a cells. In Neuro2a cells, the proteolytic processing was effectively inhibited by the proteasome inhibitors MG-132, Epoxomicin, and Lactacystin, suggesting that the ubiquitin-proteasome pathway was involved in this processing. Using MG-132, we found that the proteolytic processing changed the subcellular localization of zCaMKP-N from the nucleus to the cytosol. Accompanying this change, the cellular targets of zCaMKP-N in Neuro2a cells were significantly altered. Furthermore, we obtained evidence that the zCaMKP-N activity was markedly activated when the C-terminal domain was removed by the processing. Thus, the proteolytic processing of zCaMKP-N at the C-terminal region regulates its catalytic activity, subcellular localization and substrate targeting in vivo., (Copyright © 2011 Elsevier Inc. All rights reserved.)

We isolated cDNA clones for zebrafish Ca(2+)/calmodulin-dependent protein kinase I (zCaMKI) δ isoforms by expression screening using cDNA library from embryos at 72-h post-fertilization (hpf). There are two splice variants with different C-terminal sequences, comprising of 392 and 368 amino acids, and they are designated zCaMKIδ-L (long form) and zCaMKIδ-S (short form), respectively. Although recombinant zCaMKIδ-L and zCaMKIδ-S expressed in Escherichia coli showed essentially the same catalytic properties including substrate specificities, they showed different spatial and temporal expression. Western blotting analysis using the isoform-specific antibodies revealed that zCaMKIδ-L clearly appeared from 36hpf but zCaMKIδ-S began to appear at 60hpf and thereafter. zCaMKIδ-S was predominantly expressed in brain, while zCaMKIδ-L was widely distributed in brain, eye, ovary and especially abundantly expressed in skeletal muscle. The gene knockdown of zCaMKIδ using morpholino-based antisense oligonucleotides induced significant morphological abnormalities in zebrafish embryos. Severe phenotype of embryos exhibited short trunk, kinked tail and small heads. These phenotypes could be rescued by coinjection with the recombinant zCaMKIδ, but not with the kinase-dead mutant. These results clearly indicate that the kinase activity of zCaMKIδ plays a crucial role in the early stages in the embryogenesis of zebrafish., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Here we report a simple and useful method to detect endogenous substrates of protein kinases. When crude tissue extracts were resolved by liquid-phase isoelectric focusing (MicroRotofor) and the separated protein fractions were phosphorylated by protein kinases such as Ca(2+)/calmodulin-dependent protein kinase I or cAMP-dependent protein kinase, various proteins in the different fractions were efficiently phosphorylated. Since a higher number of substrates could significantly be detected using the resolved fractions by MicroRotofor as compared to direct analysis of the original tissue extracts, our present method will be applicable to the screening of endogenous substrates for various protein kinases., (Copyright © 2010 Elsevier Inc. All rights reserved.)

We developed a method for the detection of phosphatase activity using fluorogenic substrates after polyacrylamide gel electrophoresis. When phosphatases such as Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP), protein phosphatase 2C (PP2C), protein phosphatase 5 (PP5), and alkaline phosphatase were resolved by polyacrylamide gel electrophoresis in the absence of SDS and the gel was incubated with a fluorogenic substrate such as 4-methylumbelliferyl phosphate (MUP), all of these phosphatase activities could be detected in situ. Although 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) as well as MUP could be used as a fluorogenic substrate for an in-gel assay, MUP exhibited lower background fluorescence. Using this procedure, several fluorescent bands that correspond to endogenous phosphatases were observed after electrophoresis of various crude samples. The in-gel phosphatase assay could also be used to detect protein phosphatases resolved by SDS-polyacrylamide gel electrophoresis. In this case, however, the denaturation/renaturation process of resolved proteins was necessary for the detection of phosphatase activity. This procedure could be used for detection of renaturable protein phosphatases such as CaMKP and some other phosphatases expressed in cell extracts. The present fluorescent in-gel phosphatase assay is very useful, since no radioactive compounds or no special apparatus are required., (2009 Elsevier Inc. All rights reserved.)

Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP) dephosphorylates and regulates multifunctional Ca(2+)/calmodulin-dependent protein kinases (CaMKs). However, the biological functions of this enzyme have not been clarified in vivo. To investigate the biological significance of CaMKP during zebrafish embryogenesis, we cloned and characterized zebrafish CaMKP (zCaMKP). The isolated cDNA clone possessed an open reading frame of 1272bp encoding 424 amino acids and shared 47% and 48% amino acid identity with rat and human CaMKP, respectively. Interestingly, zCaMKP lacks the Glu cluster corresponding to residues 101-109 in the rat enzyme, and was not activated by polycations such as poly-l-lysine. The recombinant zCaMKP required Mg(2+) rather than Mn(2+) for activity. Furthermore, zCaMKP dephosphorylated CaMKIV but not phosphorylase a, alpha-casein, or extracellular signal-regulating kinase (ERK), suggesting that the enzyme regulates Ca(2+) signaling pathways in zebrafish. Cotransfection of zCaMKP with mammalian CaMKI significantly decreased phospho-CaMKI in ionomycin-stimulated 293T cells. During embryogenesis, the expression of zCaMKP increased gradually after 48h post-fertilization, as demonstrated by Western blotting using an anti-zCaMKP antibody. The knockdown of the zCaMKP gene with morpholino-based antisense oligonucleotides resulted in an increased incidence of embryos with severe morphological and cellular abnormalities, i.e., a significant increase in the number of round-shaped embryos and apoptotic cells in the whole body. A marked decrease in zCaMKP expression was observed in the antisense- but not control oligo-injected embryos. Embryonic death was rescued by coinjection with recombinant rat CaMKP but not with phosphatase-dead mutant (D194A). These results clearly show the significance of zCaMKP during zebrafish embryogenesis.

Extended-spectrum beta-lactamase (ESBL)-producing bacteria are known to be resistant to penicillins, cephalosporins, and monobactams because of their substrate specificity, and these bacteria are sensitive only to a narrow range of antimicrobial agents. The present study was undertaken to evaluate the efficacy of carbapenems and the new quinolones against ESBL-producing Escherichia coli, using a Monte Carlo simulation based on the pharmacokinetic/pharmacodynamic (PK/PD) theory. The time above MIC (TAM, %) served as the PK/PD parameter for carbapenems, with the target level set at 40%. The AUC/MIC served as the PK/PD parameter for the new quinolones, with the target level set at more than 125. In the analysis of drug sensitivity, the MIC50 of all carbapenems other than imipenem was low (0.03 microg/ml), while the MIC50 of the new quinolones was higher (1-2 microg/ml). The probability of achieving the PK/PD target with carba penems after two doses at the usual dose level, as determined by the Monte Carlo simulation, was high for each of the carbapenems tested (99.0% for biapenem, 99.60% for meropenem, and 95.03% for doripenem), except for imipenem. Among the new quinolones, the highest probability of achieving the PK/PD target was obtained with pazufloxacin (42.90%). Thus, the results of the present study have revealed that carbapenems are effective at the regular dose and can be used as the first-choice antibiotics for ESBL-producing E. coli because the resistance ratios for carbapenems are low compared to those of the new quinolones.

DNA methyltransferase 1 (Dnmt1) is an enzyme that recognizes and methylates hemimethylated CpG after DNA replication to maintain methylation patterns. Although the N-terminal region of Dnmt1 is known to interact with various proteins, such as methyl-CpG-binding protein 2 (MeCP2), the associations of protein kinases with this region have not been reported. In the present study, we found that a 110-kDa protein kinase in mouse brain could bind to the N-terminal domain of Dnmt1. This 110-kDa kinase was identified as cyclin-dependent kinase-like 5 (CDKL5) by LC-MS/MS analysis. CDKL5 and Dnmt1 were found to colocalize in nuclei and appeared to interact with each other. Catalytically active CDKL5, CDKL5(1-352), phosphorylated the N-terminal region of Dnmt1 in the presence of DNA. Considering that defects in the MeCP2 or CDKL5 genes cause Rett syndrome, we propose that the interaction between Dnmt1 and CDKL5 may contribute to the pathogenic processes of Rett syndrome.

A 30-kDa fragment of Ca(2+)/calmodulin-dependent protein kinase II (30K-CaMKII) is a constitutively active protein Ser/Thr kinase devoid of autophosphorylation activity. We have produced a chimeric enzyme of 30K-CaMKII (designated CX(40)-30K-CaMKII), in which the N-terminal 40 amino acids of Xenopus Ca(2+)/calmodulin-dependent protein kinase I (CX(40)) were fused to the N-terminal end of 30K-CaMKII. Although CX(40)-30K-CaMKII exhibited essentially the same substrate specificity as 30K-CaMKII, it underwent significant autophosphorylation. Surprisingly, its autophosphorylation site was found to be Tyr-18 within the N-terminal CX(40) region of the fusion protein, although it did not show any Tyr kinase activity toward exogenous substrates. Several lines of evidence suggested that the autophosphorylation occurred via an intramolecular mechanism. These data suggest that even typical Ser/Thr kinases such as 30K-CaMKII can phosphorylate Tyr residues under certain conditions. The possible mechanism of the Tyr residue autophosphorylation is discussed.

Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP) and its nuclear isoform CaMKP-N are unique Ser/Thr protein phosphatases that negatively regulate the Ca(2+)/calmodulin-dependent protein kinase (CaMK) cascade by dephosphorylating multifunctional CaMKI, II, and IV. However, the lack of specific inhibitors of these phosphatases has hampered studies on these enzymes in vivo. In an attempt to obtain specific inhibitors, we searched inhibitory compounds and found that Evans Blue and Chicago Sky Blue 6B served as effective inhibitors for CaMKP. These compounds also inhibited CaMKP-N, but inhibited neither protein phosphatase 2C, another member of PPM family phosphatase, nor calcineurin, a typical PPP family phosphatase. The minimum structure required for the inhibition was 1-amino-8-naphthol-4-sulfonic acid. When Neuro2a cells cotransfected with CaMKIV and CaMKP-N were treated with these compounds, the dephosphorylation of CaMKIV was strongly suppressed, suggesting that these compounds could be used as potent inhibitors of CaMKP and CaMKP-N in vivo as well as in vitro.

Objective: Alpha bungarotoxin (alpha-BTX) is a neurotoxin isolated from the venom of Bungarus multicinctus that binds specifically to the beta-subunits of nicotinic acetylcholine receptors (nAChR) on myotube membranes. The purpose of the present study was to investigate the distribution of alpha-BTX-sensitive nAChR in Hirschsprung's disease (HD) to understand the histopathological features of HD, especially the increase in acetylcholine esterase (AChE) positive nerve fibres., Methods: Confocal microscopy was used to study the expression of FITC (fluorescein isothiocyanate)-alpha-BTX, anti-synaptophysin (A-SY) antibody, and anti-neurofilament (A-NF) antibody to determine the distribution of nAChR and ganglion cell and nerve fibres in colon specimens from five cases of HD and three normal controls., Results: Quantitative assessment of the immunoreactivity of colonic muscle and colonic mucosal epithelium from an aganglionic segment of HD bowel demonstrated markedly increased nAChR compared with colonic muscle and colonic mucosal epithelium from a ganglionic segment of HD bowel and normal bowel (p < 0.0001, respectively), both of which have only a few positive nAChR. In colonic muscle from aganglionic and transitional segments of HD, there were many nAChR around hypertrophic nerve trunks identified by A-NF and A-SY staining., Conclusion: We suggest that abnormal expression of nAChR in HD might be implicated in causing gastrointestinal dysmotility because of their localization around hypertrophic nerve trunks.

Doublecortin-like protein kinase (DCLK) is a protein Ser/Thr kinase expressed in brain and believed to play crucial roles in neuronal development. To investigate the biological significance of DCLK, we isolated cDNA clones for zebrafish DCLK (zDCLK) and found that there were five splice variants of the kinase. In this study, the catalytic properties of a major isoform of zDCLK, which we designated as zDCLK1, and of an N-terminal truncated mutant retaining the kinase domain were examined by expressing them in Escherichia coli. Mutational analysis of recombinant zDCLK suggested that the kinase was activated not only by phosphorylation at Thr-576 in the activation loop but also by autophosphorylation at the other site(s) in the catalytic domain. zDCLK significantly phosphorylated protein substrates such as myelin basic protein, histones, and synapsin I. Subcellular localization of zDCLK and its N-terminal deletion mutant implicated that microtubule-association of zDCLK is mediated through N-terminal doublecortin like domain of this enzyme. Western blotting analysis and whole mount in situ hybridization revealed that zDCLK was highly expressed in brain and eyes after 24-h post fertilization. Gene knockdown of zDCLK using morpholino-based antisense oligonucleotides induced significant increase of apoptotic cells in the central nervous systems and resulted in the increase of the morphologically abnormal embryos in a dose-dependent manner. These results suggest that zDCLK may play crucial roles in the central nervous systems during the early stage of embryogenesis.

Nuclear Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP-N) is an enzyme that dephosphorylates and concomitantly downregulates multifunctional Ca2+/calmodulin-dependent protein kinases (CaMKs) in vitro. However, the functional roles of this enzyme in vivo are not well understood. To investigate the biological significance of CaMKP-N during zebrafish embryogenesis, we cloned and characterized zebrafish CaMKP-N (zCaMKP-N). Based on the nucleotide sequences in the zebrafish whole genome shotgun database, we isolated a cDNA clone for zCaMKP-N, which encoded a protein of 633 amino acid residues. Transiently expressed full-length zCaMKP-N in mouse neuroblastoma, Neuro2a cells, was found to be localized in the nucleus. In contrast, the C-terminal truncated mutant lacking RKKRRLDVLPLRR (residues 575-587) had cytoplasmic staining, suggesting that the nuclear localization signal of zCaMKP-N exists in the C-terminal region. Ionomycin treatment of CaMKIV-transfected Neuro2a cells resulted in a marked increase in the phosphorylated form of CaMKIV. However, cotransfection with zCaMKP-N significantly decreased phospho-CaMKIV in ionomycin-stimulated cells. Whole mount in situ hybridization analysis of zebrafish embryos showed that zCaMKP-N is exclusively expressed in the head and neural tube regions. Gene knockdown of zCaMKP-N using morpholino-based antisense oligonucleotides induced significant morphological abnormalities in zebrafish embryos. A number of apoptotic cells were observed in brain and spinal cord of the abnormal embryos. These results suggest that zCaMKP-N plays a crucial role in the early development of zebrafish.

Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP) is a member of the serine/threonine protein phosphatases and shares 29% sequence identity with protein phosphatase 2Calpha (PP2Calpha) in its catalytic domain. To investigate the functional domains of CaMKP, mutational analysis was carried out using various recombinant CaMKPs expressed in Escherichia coli. Analysis of N-terminal deletion mutants showed that the N-terminal region of CaMKP played important roles in the formation of the catalytically active structure of the enzyme, and a critical role in polycation stimulation. A chimera mutant, a fusion of the N-terminal domain of CaMKP and the catalytic domain of PP2Calpha, exhibited similar substrate specificity to CaMKP but not to PP2Calpha, suggesting that the N-terminal region of CaMKP is crucial for its unique substrate specificity. Point mutations at Arg-162, Asp-194, His-196, and Asp-400, highly conserved amino acid residues in the catalytic domain of PP2C family, resulted in a significant loss of phosphatase activity, indicating that these amino acid residues may play important roles in the catalytic activity of CaMKP. Although CaMKP(1-412), a C-terminal truncation mutant, retained phosphatase activity, it was found to be much less stable upon incubation at 37 degrees C than wild type CaMKP, indicating that the C-terminal region of CaMKP is important for the maintenance of the catalytically active conformation. The results suggested that the N- and C-terminal sequences of CaMKP are essential for the regulation and stability of CaMKP.

To investigate expression profiles of the entire family of protein tyrosine kinases (PTKs), we attempted to generate an antibody that detects a variety of PTKs. For production of the antibody, antigenic peptides corresponding to amino acid sequences of a highly conserved region (subdomain VIB) of PTKs were synthesized and immunized to BALB/c mice. Among various antigens, a peptide with 11 amino acids, CYVHRDLRAAN, efficiently produced a polyclonal antibody with a broad cross-reactivity to PTKs. We established a hybridoma cell line producing a monoclonal antibody, YK34, which appeared to cross-react with at least 68 PTKs in the human genome, as evidenced by its reactivity with the recombinant Src tyrosine kinases whose subdomain VIB had been replaced by those of the other PTKs. When differentiation of HL-60 cells was induced by 12-O-tetradecanoylphorbol-13-acetate, on Western blotting we observed significant changes in immunoreactive bands with YK34 in HL-60 cell extracts along with changes in the morphology of the cells. These results suggest that the YK34 antibody will be a powerful tool for analysis of a variety of cellular PTKs.

GM1a [Gal beta1-3GalNAc beta1-4(NeuAc alpha2-3)Gal beta1-4Glc beta1-1Cer] is known to support and protect neuronal functions. However, we report that alpha-linolenic acid-containing GM1a (C18:3-GM1a), which was prepared using the reverse hydrolysis reaction of sphingolipid ceramide N-deacylase, induced apoptosis in neuronal cells. Intranucleosomal DNA fragmentation, chromatin condensation, and caspase activation, all typical features of apoptosis, were observed when mouse neuroblastoma Neuro2a cells were cultured with C18:3-GM1a but not GM1a containing stearic acid (C18:0) or oleic acid (C18:1). The phenotype of Neuro2a cells induced by C18:3-GM1a was similar to that evoked by lyso-GM1a. However, lyso-GM1a caused a complete disruption of lipid microdomains of Neuro2a cells and hemolysis of sheep erythrocytes, whereas C18:3-GM1a did neither. C18:3-GM1a, but not lyso-GM1a, was found to be abundant in lipid microdomains after the removal of loosely bound GM1a by BSA. The activation of stress-activated protein kinase/c-Jun N-terminal kinase in Neuro2a cells was observed with lyso-GM1a but not C18:3-GM1a. These results indicate that the mechanism of apoptosis induced by C18:3-GM1a is distinct from that caused by lyso-GM1a. This study also clearly shows that fatty acid composition of gangliosides significantly affected their pharmacological activities when added to the cell cultures and suggests why naturally occurring gangliosides do not possess polyunsaturated fatty acids as a major constituent.

Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP) is a unique protein phosphatase that specifically dephosphorylates and regulates multifunctional Ca(2+)/calmodulin-dependent protein kinases (CaMKs). To clarify the physiological significance of CaMKP, we identified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and fructose bisphosphate aldolase as major binding partners of CaMKP in a soluble fraction of rat brain using the two-dimensional far-Western blotting technique, in conjunction with peptide mass fingerprinting analysis. We analyzed the affinities of these interactions. Wild type CaMKP-glutathione S-transferase (GST) associated with GAPDH in a GST pull-down assay. Deletion analysis suggested that the N-terminal side of the catalytic domain of CaMKP was responsible for the binding to GAPDH. Further, anti-CaMKP antibody coimmunoprecipitated GAPDH in a rat brain extract. GAPDH was phosphorylated by CaMKI or CaMKIV in vitro; however, when CaMKP coexisted, the phosphorylation was markedly attenuated. Under these conditions, CaMKP significantly dephosphorylated CaMKI and CaMKIV, which had been phosphorylated by CaMK kinase, whereas it did not dephosphorylate the previously phosphorylated GAPDH. The results suggest that CaMKP regulates the phosphorylation level of GAPDH in the CaMKP-GAPDH complex by dephosphorylating and deactivating CaMKs that are responsible for the phosphorylation of GAPDH.

We studied the antimicrobial susceptibility of AmpC beta-lactamase-producing Escherichia coli isolates collected at ten medical institutions in the Kinki area of Japan during a 6-month period (November 2002 through April 2003). Of 2845 E. coli isolates tested, 29 (1.0%) showed a minimum inhibitory concentration (MIC) for cefazolin of more than 8 microg/ml and were three-dimensional extract test positive. In standard inoculum susceptibility tests against these 29 strains, the MIC90s for the four carbapenems tested ranged from 0.06 microg/ml to 0.5 microg/ml, and these compounds were more active than the other beta-lactams, with meropenem being the most active. The MIC90s for beta-lactams, except carbapenems, ranged from 4 microg/ml to 32 microg/ml, with cefepime being the most active. In high inoculum susceptibility tests against these strains, the MIC90s for the four carbapenems and cefepime were 8 microg/ml or less, and these compounds were more active than other beta-lactams. The MIC90s for beta-lactams, except carbapenems and cefepime, were 32 microg/ml or more. The MIC90s for the five quinolones tested ranged from 4 microg/ml to 16 microg/ml, and the order of increasing susceptibility was ciprofloxacin > levofloxacin, gatifloxacin and pazufloxacin > prulifloxacin.

Background: Transforming growth factor-beta (TGF-beta) has been implicated in the development of renal fibrosis induced by unilateral ureteral obstruction (UUO). However, there is little information on signaling pathways mediating TGF-beta activity involved in molecular and cellular events leading to renal fibrosis induced by UUO. In this study, we sought to determine whether Smad3, a major signaling component of TGF-beta, mediated renal fibrosis induced by UUO., Methods: Renal fibrosis, inflammation, and apoptosis induced by UUO were macroscopically and histologically compared between wild-type mice and Smad3 null mice., Results: Gross appearance of the kidney after UUO showed relatively intact kidney in Smad3 null mice [Smad3(-/-) mice] when compared with that of wild-type mice [Smad3(+/+) mice]. Renal interstitial fibrosis based on the interstitial area stained with Aniline-blue or Sirius red solution was significantly attenuated in the obstructed kidney of Smad3(-/-) mice when compared with that of Smad3(+/+) mice. Deposition of type I and type III collagens were also significantly reduced in the obstructed kidney of Smad3(-/-) mice. In addition, the numbers of myofibroblasts, macrophages, and CD4/CD8 T cells infiltrated into the kidney after UUO were significantly attenuated in the obstructed kidney of Smad3(-/-) mice when compared with that of Smad3(+/+) mice. Furthermore, terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) staining after UUO showed significantly reduced number of tubular apoptotic cells in the obstructed kidney of Smad3(-/-) mice when compared with that of Smad3(+/+) mice. Endogenous Smad pathway was activated in the obstructed kidney after UUO in wild-type mice as judged by the increase of phosphorylated Smad2 or phosphorylated Smad2/3-positive cells in renal interstitial area., Conclusion: Smad3 deficiency attenuated renal fibrosis, inflammation, and apoptosis after UUO, suggesting that Smad3 was a key molecule mediating TGF-beta activity leading to real fibrosis after UUO.

To explore the protein kinase family enzymes expressed in cells, we attempted to generate antibodies that could detect a wide variety of protein kinases. For the production of such antibodies, synthetic peptides corresponding to amino acid sequences of a highly conserved subdomain (subdomain VIB) of the protein kinase family were used for immunization. Among the various peptide antigens, a peptide with 16 amino acids, CVVHRDLKPENLLLAS, effectively produced polyclonal antibodies with broad cross-reactivities to protein kinases. Two monoclonal antibodies, designated M8C and M1C, detected a variety of protein kinases such as calmodulin-dependent protein kinase II, calmodulin-dependent protein kinase IV, cAMP-dependent protein kinase, and mitogen-activated protein kinases, on Western blotting. The antibodies also immunoprecipitated various protein kinases in cell extracts. Furthermore, these antibodies could be used for detection of positive clones in the expression cloning of various protein kinases. Among 39 positive clones obtained from mouse brain cDNA library, 36 clones were identified as cDNA clones for various known and novel protein serine/threonine kinases, suggesting that the antibodies reacted highly specifically with various protein kinases. These results indicate that the present monoclonal antibodies directed to multiple protein kinases will be a powerful tool for the detection of a variety of known and novel protein kinases in cells.

Fas ligand (FasL) has been implicated in cytotoxic T lymphocyte (CTL)- and natural killer (NK) cell-mediated cytotoxicity. In the present study, we investigated the localization of FasL in murine CTL and NK cells. Immunocytochemical staining showed that FasL was stored in cytoplasmic granules of CD8+ CTL clones and in vivo activated CTL and NK cells, where perforin and granzyme A also resided. Immunoelectron microscopy revealed that FasL was localized on outer membrane of the cytoplasmic granules, while perforin was localized in internal vesicles. Western blot analysis showed that the membrane-type FasL of 40 kDa was stored in CD8+ CTL clones but not in CD4+ CTL clones. By utilizing a granule exocytosis inhibitor (TN16), we demonstrated that FasL translocated onto cell surface upon degranulation of anti-CD3-stimulated CD8+ CTL clones. Moreover, TN16 markedly inhibited the FasL-mediated cytotoxicity by CD8+ T cell clones and NK cells. These results suggested a substantial contribution of FasL to granule exocytosis-mediated target cell lysis by CD8+ CTL and NK cells.

Lysosphingolipids, which lack the fatty acid moiety of sphingolipids, are known to be accumulated in some variants of sphingolipid storage diseases. Here, we report that lysosphingolipids with naturally occurring stereochemical configurations induce apoptosis in mouse neuroblastoma Neuro2a cells. The intracellular dehydrogenase activity and [3H]thymidine incorporation of Neuro2a cells were strongly suppressed by the addition of lysosphingolipids in a dose-dependent manner, whereas the parental sphingolipids had no effect. Intranucleosomal DNA fragmentation, chromatin condensation, and phosphatidylserine externalization, which are typical features of apoptosis, were observed when the cells were cultured with 40-80 microM of lysosphingolipids for 24-48 h in the presence of 5% fetal calf serum. Activation of caspase-3-like enzyme occurred after addition of lysosphingolipids followed by incubation at 37 degrees C for 24 h. The addition of an inhibitor of caspases, ZVAD-fmk, to the Neuro2a cell culture completely inhibited the elevation of caspase-3 activity but not the DNA fragmentation. These results may indicate that a caspase-3 independent signaling pathway is involved in the lysosphingolipid-induced apoptosis and suggest that accumulation of lysosphingolipids, but not parental sphingolipids, triggers the apoptotic cascade in neuronal cells of patients with sphingolipidoses.

Sphingosylphosphocholine, an N-deacylated derivative of sphingomyelin, has been found to be involved in many cellular events. This paper describes a new method for preparation of a D-erythro-sphingosylphosphocholine, which is naturally occurring but difficult to prepare by chemical methods, using marine bacteria as a biocatalyst. When cultured with Shewanella alga NS-589 in synthetic medium, sphingomyelin was found to be efficiently converted by sphingomyelin deacylase to sphingosylphosphocholine. Sphingosylphosphocholine was purified with a high yield from the culture supernatant and identified to be a D-erythro-(2S,3R)-isomer containing d18:1 sphingenine as a long-chain base by fast atom bombardment mass spectrometry and NMR analyses.