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AIM: Glucokinase (GK) in pancreatic beta cells is thought to be involved in insulin secretion and glucose homeostasis. This study investigates whether the long-acting agonist of the glucagon-like peptide 1, namely exendin-4, mediates stimulatory effects on GK gene expression through the Ca(2+)/calmodulin (CaM)-dependent protein kinase (CaMK) cascade. METHODS: GK expression was examined by real-time PCR, western blot analysis and reporter gene assay in rat insulin-secreting INS-1 cells incubated with exendin-4. CaMKIV activity was assessed by detection of activation loop phosphorylation (Thr(196)) of CaMKIV. We investigated the effect of the constitutively active form (CaMKIVc) of CaMKIV on GK promoter activity. RESULTS: Increased expression level of GK protein was noted in response to rising concentrations of exendin-4 with maximum induction at 10 nM. Real-time PCR analysis showed a significant increase in the amount of GK mRNA in response to rising concentrations of exendin-4. Exendin-4 also stimulated GK promoter activity but failed to do so in the presence of STO-609, a CaMKK inhibitor. This result is consistent with the observations that the upregulation of CaMKIV phosphorylation (at Thr(196)) peaked after 15 min of exposure to exendin-4 and that CaMKIVc enhanced or upregulated GK promoter activity in INS-1 cells. Furthermore, STO-609 significantly suppressed the exendin-4 - upregulated the expression of the GK protein. CONCLUSION: Activation of the CaMKK/CaMKIV cascade might be required for exendin-4-induced GK gene transcription, indicating that exendin-4 plays an important role in insulin secretion in pancreatic beta cells.

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In order to consider the total atmospheric loadings of the PAHs (polycyclic aromatic hydrocarbons) from traffic activities, the emission factors of PAHs were estimated and from the obtained emission factors and vehicle transportation statistics, total atmospheric loadings were integrated and the loadings into the water body were estimated on a regional scale. The atmospheric concentration of PAHs was measured at the roadside of a road with heavy traffic in the Hiroshima area in Japan. The samplings were conducted in summer and winter. Atmospheric particulate matters (fine particle, 0.6-7 microm; coarse particle, over 7 microm) and their PAH concentration were measured. Also, four major emission sources (gasoline and diesel vehicle emissions, tire and asphalt debris) were assumed for vehicle transportation activities, the chemical mass balance method was applied and the source partitioning at the roadside was estimated. Furthermore, the dispersion of atmospheric particles from the vehicles was modelled and the emission factors of the sources were determined by the comparison to the chemical mass balance results. Based on emission factors derived from the modelling, an atmospheric dispersion model of nationwide scale (National Institute of Advanced Industrial Science and Technology - Atmospheric Dispersion Model for Exposure and Risk assessment) was applied, and the atmospheric concentration and loading to the ground were calculated for the Hiroshima Bay watershed area.

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We have previously demonstrated that the alpha isoform of Ca(2+)/calmodulin-dependent protein kinase kinase (CaM-KKalpha) is strictly regulated by an autoinhibitory mechanism and activated by the binding of Ca(2+)/CaM [Tokumitsu, H., Muramatsu, M., Ikura, M., and Kobayashi, R. (2000) J. Biol. Chem. 275, 20090-20095]. In this study, we find that rat brain extract contains Ca(2+)/CaM-independent CaM-KK activity. This result is consistent with an enhanced Ca(2+)/CaM-independent activity (60-70% of total activity) observed with the recombinant CaM-KKbeta isoform. By using various truncation mutants of CaM-KKbeta, we have identified a region of 23 amino acids (residues 129-151) located at the N-terminus of the catalytic domain as an important regulatory element of the autonomous activity. A CaM-KKbeta deletion mutant of this domain shows a significant increase of Ca(2+)/CaM dependency for the CaM-KK activity as well as for the autophosphorylation activity. The activities of CaM-KKalpha and CaM-KKbeta chimera, in which autoinhibitory sequences were replaced by each other, were completely dependent on Ca(2+)/CaM, suggesting that the autoinhibitory regions of CaM-KKalpha and CaM-KKbeta are functional. These results establish for the first time that residues 129-151 of CaM-KKbeta participate in the release of the autoinhibitory domain from its catalytic core, resulting in generation of autonomous activity.

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Calmodulin (CaM) is a ubiquitous calcium (Ca(2+)) sensor which binds and regulates protein serine/threonine kinases along with many other proteins in a Ca(2+)-dependent manner. For this multi-functionality, conformational plasticity is essential; however, the nature and magnitude of CaM's plasticity still remains largely undetermined. Here, we present the 1.8 A resolution crystal structure of Ca(2+)/CaM, complexed with the 27-residue synthetic peptide corresponding to the CaM-binding domain of the nematode Caenorhabditis elegans Ca(2+)/CaM-dependent kinase kinase (CaMKK). The peptide bound in this crystal structure is a homologue of the previously NMR-derived complex with rat CaMKK, but benefits from improved structural resolution. Careful comparison of the present structure to previous crystal structures of CaM complexed with unrelated peptides derived from myosin light chain kinase and CaM kinase II, allow a quantitative analysis of the differences in the relative orientation of the N and C-terminal domains of CaM, defined as a screw axis rotation angle ranging from 156 degrees to 196 degrees. The principal differences in CaM interaction with various peptides are associated with the N-terminal domain of CaM. Unlike the C-terminal domain, which remains unchanged internally, the N-terminal domain of CaM displays significant differences in the EF-hand helix orientation between this and other CaM structures. Three hydrogen bonds between CaM and the peptide (E87-R336, E87-T339 and K75-T339) along with two salt bridges (E11-R349 and E114-K334) are the most probable determinants for the binding direction of the CaMKK peptide to CaM.

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To elucidate the molecular mechanism involved in the suppression of keloids and hypertrophic scars by tranilast, we investigated the target protein of tranilast in bovine skin and aorta. A specific tranilast-binding protein was isolated from both tissues by drug affinity chromatography and was identified as 36-kDa microfibril-associated glycoprotein (36-kDa MAGP). Binding of 36-kDa MAGP to tranilast seemed to be specific since 36-kDa MAGP could be eluted from the drug affinity column by tranilast itself and also binding of 36-kDa MAGP to other anti-allergy drugs (amlexanox and cromolyn) is significantly weaker than that to tranilast. Light and electron microscopic immunohistochemistry detected the protein at the periphery of elastic fibers in normal human skin. In hypertrophic scar tissue, however, 36-kDa MAGP was located on small bundles of microfibrils. These findings provide support for the concept that elastogenesis occurs in scar tissue and 36-kDa MAGP might be one of the targets for tranilast.

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Ca(2+)/calmodulin-dependent protein kinase kinase (CaM-KK) is a novel member of the CaM kinase family, which specifically phosphorylates and activates CaM kinase I and IV. In this study, we characterized the CaM-binding peptide of alphaCaM-KK (residues 438-463), which suppressed the activity of constitutively active CaM-KK (84-434) in the absence of Ca(2+)/CaM but competitively with ATP. Truncation and site-directed mutagenesis of the CaM-binding region in CaM-KK reveal that Ile(441) is essential for autoinhibition of CaM-KK. Furthermore, CaM-KK chimera mutants containing the CaM-binding sequence of either myosin light chain kinases or CaM kinase II located C-terminal of Leu(440), exhibited enhanced Ca(2+)/CaM-independent activity (60% of total activity). Although the CaM-binding domains of myosin light chain kinases and CaM kinase II bind to the N- and C-terminal domains of CaM in the opposite orientation to CaM-KK (Osawa, M., Tokumitsu, H., Swindells, M. B., Kurihara, H., Orita, M., Shibanuma, T., Furuya, T., and Ikura, M. (1999) Nat. Struct. Biol. 6, 819-824), the chimeric CaM-KKs containing Ile(441) remained Ca(2+)/CaM-dependent. This result demonstrates that the orientation of the CaM binding is not critical for relief of CaM-KK autoinhibition. However, the requirement of Ile(441) for autoinhibition, which is located at the -3 position from the N-terminal anchoring residue (Trp(444)) to CaM, accounts for the opposite orientation of CaM binding of CaM-KK compared with other CaM kinases.

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The potential of gadolinium neutron-capture therapy (Gd-NCT) for cancer was evaluated using chitosan nanoparticles as a novel gadolinium device. The nanoparticles, incorporating 1200 microg of natural gadolinium, were administered intratumorally twice in mice bearing subcutaneous B16F10 melanoma. The thermal neutron irradiation was performed for the tumor site, with the fluence of 6. 32x10(12) neutrons/cm(2), 8 h after the second gadolinium administration. After the irradiation, the tumor growth in the nanoparticle-administered group was significantly suppressed compared to that in the gadopentetate solution-administered group, despite radioresistance of melanoma and the smaller Gd dose than that administered in past Gd-NCT trials. This study demonstrated the potential usefulness of Gd-NCT using gadolinium-loaded nanoparticles.

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The structure of calcium-bound calmodulin (Ca2+/CaM) complexed with a 26-residue peptide, corresponding to the CaM-binding domain of rat Ca2+/CaM-dependent protein kinase kinase (CaMKK), has been determined by NMR spectroscopy. In this complex, the CaMKK peptide forms a fold comprising an alpha-helix and a hairpin-like loop whose C-terminus folds back on itself. The binding orientation of this CaMKK peptide by the two CaM domains is opposite to that observed in all other CaM-target complexes determined so far. The N- and C-terminal hydrophobic pockets of Ca2+/CaM anchor Trp 444 and Phe 459 of the CaMKK peptide, respectively. This 14-residue separation between two key hydrophobic groups is also unique among previously determined CaM complexes. The present structure represents a new and distinct class of Ca2+/CaM target recognition that may be shared by other Ca2+/CaM-stimulated proteins.

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Biodegradable gadopentetic acid (Gd-DTPA)-loaded chitosan microparticles (Gd-microCPs) were prepared as a device for gadolinium neutron-capture therapy (Gd-NCT) by a novel emulsion-droplet coalescence technique: a water-in-oil (w/o) emulsion A containing chitosan and Gd-DTPA in droplets and a w/o emulsion B containing NaOH in droplets were mixed and stirred to solidify chitosan as a result of collision and coalescence between droplets of each emulsion. Gd-microCPs prepared by using 100% deacetylated chitosan in 25% Gd-DTPA solution were 4.1 microns (non-lyophilized) and 3.3 microns (lyophilized) in mass median diameter, and were 3.4% in gadolinium content, corresponding to 11.7% as Gd-DTPA. The particle size and gadolinium content of Gd-microCPs were not affected by Gd-DTPA concentration in the chitosan medium. However, the deacetylation degree of chitosan influenced the particle size; as the deacetylation degree of chitosan decreased, the particle size increased. The incorporated Gd-DTPA was not released entirely from Gd-microCPs in an isotonic phosphate buffered saline solution despite the high water-solubility of Gd-DTPA (less than 0.8% with every type of Gd-microCPs). These results indicated that ion-complex formation might be contributable to incorporation of Gd-DTPA. As a preliminary study, it was confirmed that the loss of gamma-ray emission by gadolinium-loading in microparticle was negligible in the thermal neutron irradiation test in vitro. These results suggested that Gd-microCPs could be a useful device for intratumoral injection into solid tumor on Gd-NCT.

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We have recently demonstrated that Caenorhabditis elegans Ca(2+)/calmodulin-dependent protein kinase kinase (CeCaM-KK) can activate mammalian CaM-kinase IV in vitro (Tokumitsu, H., Takahashi, N., Eto, K., Yano, S., Soderling, T.R., and Muramatsu, M. (1999) J. Biol. Chem. 274, 15803-15810). In the present study, we have identified and cloned a target CaM-kinase for CaM-KK in C. elegans, CeCaM-kinase I (CeCaM-KI), which has approximately 60% identity to mammalian CaM-KI. CeCaM-KI has 348 amino acid residues with an apparent molecular mass of 40 kDa, which is activated by CeCaM-KK through phosphorylation of Thr(179) in a Ca(2+)/CaM-dependent manner, resulting in a 30-fold decrease in the K(m) of CeCaM-KI for its peptide substrate. Unlike mammalian CaM-KI, CeCaM-KI is mainly localized in the nucleus of transfected cells because the NH(2)-terminal six residues ((2)PLFKRR(7)) contain a functional nuclear localization signal. We have also demonstrated that CeCaM-KK and CeCaM-KI reconstituted a signaling pathway that mediates Ca(2+)-dependent phosphorylation of cAMP response element-binding protein (CREB) and CRE-dependent transcriptional activation in transfected cells, consistent with nuclear localization of CeCaM-KI. These results suggest that the CaM-KK/CaM-KI cascade is conserved in C. elegans and is functionally operated both in vitro and in intact cells, and it may be involved in Ca(2+)-dependent nuclear events such as transcriptional activation through phosphorylation of CREB.

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Mammalian Ca2+/CaM-dependent protein kinase kinase (CaM-KK) has been identified and cloned as an activator for two kinases, CaM kinase I (CaM-KI) and CaM kinase IV (CaM-KIV), and a recent report (Yano, S., Tokumitsu, H., and Soderling, T. R. (1998) Nature 396, 584-587) demonstrates that CaM-KK can also activate and phosphorylate protein kinase B (PKB). In this study, we identify a CaM-KK from Caenorhabditis elegans, and comparison of its sequence with the mammalian CaM-KK alpha and beta shows a unique Arg-Pro (RP)-rich insert in their catalytic domains relative to other protein kinases. Deletion of the RP-domain resulted in complete loss of CaM-KIV activation activity and physical interaction of CaM-KK with glutathione S-transferase-CaM-KIV (T196A). However, CaM-KK autophosphorylation and phosphorylation of a synthetic peptide substrate were normal in the RP-domain mutant. Site-directed mutagenesis of three conserved Arg in the RP- domain of CaM-KK confirmed that these positive charges are important for CaM-KIV activation. The RP- domain deletion mutant also failed to fully activate and phosphorylate CaM-KI, but this mutant was indistinguishable from wild-type CaM-KK for the phosphorylation and activation of PKB. These results indicate that the RP-domain in CaM-KK is critical for recognition of downstream CaM-kinases but not for its catalytic activity (i.e. autophosphorylation) and PKB activation.

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PURPOSE: The gadopentetic acid (Gd-DTPA)-loaded chitosan nanoparticles (Gd-nanoCPs) were prepared for gadolinium neutron-capture therapy (Gd-NCT) and characterized and evaluated as a device for intratumoral (i.t.) injection. METHODS: Gd-nanoCPs were prepared by a novel emulsion-droplet coalescence technique. The effects of the deacetylation degree of chitosan and Gd-DTPA concentration in chitosan medium on the particle size and the gadolinium content in Gd-nanoCPs were examined. In vitro Gd-DTPA release from Gd-nanoCPs was evaluated using an isotonic phosphate-buffered saline solution (PBS, pH 7.4) and human plasma. In vivo Gd-DTPA retention in the tumor after i.t. injection of Gd-nanoCPs was estimated on mice bearing s.c. B16F10 melanoma. RESULTS: Gd-nanoCPs with the highest Gd content, which were obtained using 100% deacetylated chitosan in 15% Gd-DTPA aqueous solution, were 452 nm in diameter and 45% in Gd-DTPA content. A lower deacetylation degree of chitosan led to an increase in particle size and a decrease in Gd-DTPA content in Gd-nanoCPs. As Gd-DTPA concentration in the chitosan solution increased, Gd-DTPA content in Gd-nanoCPs increased but the particle size did not vary. Gd-DTPA loaded to Gd-nanoCPs was hardly released over 7 days in PBS (1.8%) despite the high water solubility of Gd-DTPA. In contrast, 91% of Gd-DTPA was released in plasma over 24 hours. When Gd-nanoCPs were i.t. injected, 92% of Gd-DTPA injected effectually without outflow was held in the tumor tissue for 24 hours, which was different from the case of gadopentetate solution injection (only 1.2%). CONCLUSIONS: Gd-nanoCPs highly incorporating Gd-DTPA were successfully prepared by the emulsion-droplet coalescence technique. Their releasing properties and their ability for long-term retention of Gd-DTPA in the tumor indicated that Gd-nanoCPs might be useful as an i.t. injectable device for Gd-NCT.

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The protection against apoptosis provided by growth factors in several cell lines is due to stimulation of the phosphatidylinositol-3-OH kinase (PI(3)K) pathway, which results in activation of protein kinase B (PKB; also known as c-Akt and Rac) and phosphorylation and sequestration to protein 14-3-3 of the proapoptotic Bcl-2-family member BAD. A modest increase in intracellular Ca2+ concentration also promotes survival of some cultured neurons through a pathway that requires calmodulin but is independent of PI(3)K and the MAP kinases. Here we report that Ca2+/calmodulin-dependent protein kinase kinase (CaM-KK) activates PKB directly, resulting in phosphorylation of BAD on serine residue 136 and the interaction of BAD with protein 14-3-3. Serum withdrawal induced a three- to fourfold increase in cell death of NG108 neuroblastoma cells, and this apoptosis was largely blocked by increasing the intracellular Ca2+ concentration with NMDA (N-methyl-D-aspartate) or KCl or by transfection with constitutively active CaM-KK. The effect of NMDA on cell survival was blocked by transfection with dominant-negative forms of CaM-KK or PKB. These results identify a Ca2+-triggered signalling cascade in which CaM-KK activates PKB, which in turn phosphorylates BAD and protects cells from apoptosis.

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A combination study of cerebrospinal fluid scintigraphy and nasal pledget counts was performed using 37 MBq of 111In-DTPA in 12 patients with suspected rhinorrhea. A pledget was inserted and dwelled in each nasal cavity for 6 hours, with the patient prone during at least 30 minutes. A total of 18 studies was implemented and nasal pledget counting method successfully diagnosed all of CSF rhinorrhea. Diagnosis was possible when pledget counts were greater than 1 kcpm. In patients with persistent, intermittent and occult/no nasal discharge, rhinorrhea was found in 100% (5/5), 60% (3/5), 25% (2/8), respectively. Two cases only exhibited positive scintigraphy. MRI or CT cisternography should be first performed in patients with persistent discharge, but in patients with intermittent/occult discharge pledget counting method might take priority of other diagnostic modalities. In conclusion, nasal pledget counting method is a simple and useful tool for detecting rhinorrhea.

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A novel protein kinase related to the C. elegans serine/threonine kinase UNC-51 was cloned from mouse. The UNC-51-Like Kinase (ULK)1 is encoded by a cDNA of 1051 amino acids with calculated MW of 113 kDa. Comparison of the ULK1 and UNC-51 shows the highest conservation in the amino-terminal kinase domain, which is followed by a proline/serine-rich (PS) domain and a conserved carboxyl-terminal (C) domain. ULK1 mRNA is expressed in various tissues, and is mapped to mouse chromosome 5F and rat chromosome 12q16.3, by fluorescent in situ hybridization. HA-tagged ULK1 is expressed as a protein of approximately 150 kDa in COS7 cells and is auto-phosphorylated in vitro in its PS domain. We propose that ULK1, UNC-51 and a yeast protein kinase Apg1p comprise a novel subfamily of protein kinase, which is structurally conserved among eukaryotes.

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A 28-year-old woman was referred to us to undergo 131I therapy who had multiple pulmonary metastases from papillary thyroid carcinoma after total thyroidectomy. There was no increased accumulation of a tracer in the pulmonary metastatic foci on whole-body scanning using a 111 MBq diagnostic dose of 131I. However, the pulmonary metastases were gradually decreased in size, and then clearly reduced 8 months after the start of TSH suppression therapy, which was maintained by T3 instead of T4 to bring down the serum TSH level below 0.1 microU/ml. Reduction rates of the foci were 33-76% on chest X-ray. The reduction was confirmed and no new lesions were found on the serial CT scans. Serum thyroglobulin level was lowered 80 to 25 ng/ml by this suppression therapy and progression of disease was not observed under a 54 months' T3 treatment. Thus, TSH suppression therapy might improve survival of patients with differentiated thyroid cancer.

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The stability of tert-butoxycarbonyl-Tyr-Leu-Val-CH2Cl (YLV) with inhibitory effect on human leukocyte elastase was investigated in aqueous solution, alpha-chymotrypsin solution and biological media. In all cases studied here, the degradation was observed as a pseudo-first order reaction. The half-life for the degradation of YLV in an aqueous solution of pH 7.4 at 37 degrees C was 35.9 h. YLV was most stable at about pH 3.8-5.8 and the effect of temperature was explained by the Arrhenius equation. The activation energies of the degradation in aqueous solutions at pH 2.0, 4.8, and 7.4 were 24.6, 22.1 and 23.4 kcal/mol, respectively. The degradation products in aqueous solution were analyzed by HPLC-MS and were estimated as Boc-Tyr-Leu-Val-CH2OH at pH 7.4 and H2N-Tyr-Leu-Val-CH2Cl at pH 2.0. In a bovine pancreas alpha-chymotrypsin solution at 37 degrees C, the half-life of YLV was 15 min at 25.6 micrograms/ml of alpha-chymotrypsin solution. In the rat plasma, the half-life of YLV was 42.4 min (YLV 26.7 micrograms/ml plasma), and in rat liver, lung and spleen homogenates, the degradation rate constants of YLV were 37.6, 10.3 and 23.5 times larger than that in plasma solution, respectively (all fluids containing 5 mg protein/ml). YLV was less stable than nafarelin acetate, secretin, adrenocorticotropic hormone (ACTH) and gonadorelin in an aqueous solution of pH 7.4.

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We recently cloned a calmodulin-dependent protein kinase kinase (CaM-KK) which phosphorylates and activates CaM-KI and CaM-KIV [Tokumitsu, H., Enslen, H., and Soderling, T. R. (1995) J. Biol. Chem. 270, 19320-19324]. In the present study, we have identified its regulatory CaM-binding and autoinhibitory domains (CBD and AID, respectively) using a series of COOH-terminal truncations and site-directed mutants expressed in COS-7 cells. Truncation mutant CaM-KK1-463 activated CaM-KIV and bound CaM similar to wild-type enzyme (CaM-KK1-505); CaM-KK1-448 did not bind CaM and was largely inactive; and CaM-KK1-434 also did not bind CaM but activated a CaM-independent mutant of CaM-KIV in the absence of Ca2+/CaM. Substitution of triple negative charges (Asp) at positions 455RKR, 448ILV, or 443SWT blocked CaM binding and suppressed by 70-90% CaM-KK activities. Mutants 438VKL and 435KNS to DDD exhibited partial Ca2+/CaM-independent activities. These results identify overlapping AID and CBD between residues 430 and 460 in CaM-KK, similar to other CaM-Ks. Consistent with this assignment, the synthetic peptide corresponding to residues 438-463 bound CaM in a Ca2+-dependent manner with a Kd in the low nanomolar range. Furthermore, phosphorylation by cAMP-kinase of Ser458 at the COOH-terminus of the CBD in CaM-KK, which suppresses subsequent CaM binding [Wayman, G., Tokumitsu, H., and Soderling, T. R. (1997) J. Biol. Chem. 272, 16073-16076], was blocked by prior binding of Ca2+/CaM to CaM-KK.

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The calmodulin-dependent kinase (CaM-K) cascade, a Ca2+-triggered system involving phosphorylation and activation of CaM-KI and CaM-KIV by CaM kinase kinase (CaM-KK), regulates transcription through direct phosphorylation of transcription factors such as cAMP response element-binding protein. We have shown previously that activated CaM-KIV can activate the mitogen-activated protein kinases (Enslen, H., Tokumitsu, H., Stork, P. J. S., Davis, R. J., and Soderling, T. R. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 10803-10808), and the present paper describes a novel regulatory cross-talk between cAMP kinase (PKA) and CaM-KK. PKA gave rapid phosphorylation in vitro and in cells of recombinant CaM-KK, resulting in 50-75% inhibition of CaM-KK activity, part of which was due to suppression of CaM-binding by phosphorylation of Ser458 in the CaM-binding domain. However, the Ser458 --> Ala mutant, or a truncation mutant in which the CaM-binding and autoinhibitory domains were deleted, was still partially suppressed by PKA-mediated phosphorylation. The second inhibitory site was identified as Thr108 by site-specific mutagenesis. Treatments of COS-7, PC12, hippocampal, or Jurkat cells with the PKA activators forskolin or isoproterenol gave 30-90% inhibition of either endogenous or transfected CaM-KK and/or CaM-KIV activities. These results demonstrate that the CaM kinase cascade is negatively regulated in cells by the cAMP/PKA pathway.

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