Characterization of in vitro biotransformation of the new oral anticoagulants, the factor VIIa inhibitors AS1927819-00 and AS1932804-00.

We recently developed a prodrug (AS1932804-00, CMP) of the novel FVIIa inhibitor AS1924269-00, which possesses a carbamate amidine backbone. In addition, we developed another type of prodrug (AS1927819-00, OXP) with an oxime amidine backbone. In this study, we investigated the efficiency of conversion of these novel FVIIa prodrugs to their active forms by evaluating the production of the active form in vitro by using microsomes, mitochondria, and cryopreserved hepatocytes, and compared it with the in vivo conversion mechanisms of the prodrugs (oxime amidine vs. carbamate amidine). We observed that OXP and CMP showed improved oral absorption, and the efficiency of conversion of CMP to the active form was higher than that of OXP. The in vivo rate of conversion of OXP to its active form was low in rats, and compared to liver microsomes and mitochondria, cryopreserved hepatocytes supplemented with serum and coenzymes were an appropriate metabolic test tool. On the other hand, the efficiency of conversion of CMP to its active from could be appropriately evaluated using small intestinal microsomes. The development of a prodrug can be optimized when information about the stability of carboxylic acid esters in the presence of serum esterases, membrane permeability of intermediate forms, and differential tissue specificity to metabolic activities for carbamate and oxime backbones of amidine can be obtained.

Pharmacokinetics of the amidine prodrug of a novel oral anticoagulant factor VIIa inhibitor (AS1924269-00) in rats.

AS1924269-00 is a promising orally applicable anticoagulant that inhibits FVIIa but has very low oral absorption. Therefore, in this study, we aimed to develop a prodrug of AS1924269-00, which possesses a carbamate-added amidine functional group, with high membrane permeability. We investigated the pharmacokinetics of the carbamate-type prodrug of AS1924269-00 in rats. The Caco-2 cell monolayer was used as an in vitro model and in parallel an artificial membrane permeability assay (PAMPA) was performed to examine the transport mechanisms of the prodrug. The bioavailability of the active form was determined to be only 0.3% in rats, but the oral absorption of the prodrug was markedly improved, and its bioavailability was 36%. Our in vivo result was consistent with the finding that compared to AS1924269-00, the prodrug showed favorable permeability in Caco-2 cells and PAMPA. We introduced carbamate into the amidine functional group of the FVIIa inhibitor, which possesses the amidine backbone, and converted it to a prodrug using carboxylic acid ethyl ester. This novel prodrug had favorable absorption and membrane permeability in vivo and in vitro. Thus, we suggest a clinical application of the carbamate-added amidine prodrug of the FVIIa inhibitor.

Oral administration of milk fermented with Lactococcus lactis subsp. cremoris FC protects mice against influenza virus infection.

AIMS: To evaluate the protective effects of oral administration of milk fermented with a Lactococcus strain against influenza virus (IFV) infection in a mouse model. METHODS AND RESULTS: Milk fermented with exopolysaccharide-producing Lactococcus lactis subsp. cremoris (L. cremoris) FC was orally administered to BALB/c mice for 12 days. Mice were intranasally infected with IFV A/New Caledonia/20/99 (H1N1) on day 8, and survival was determined for 14 days after IFV infection. Survival rate and body weight loss after IFV infection in the L. cremoris FC fermented milk-administered group were significantly improved compared with those in the control group. In the unfermented milk-administered group, survival rate was not improved, whereas body weight loss was slightly improved compared with that in the control group. The mean virus titre in the lung of the L. cremoris FC fermented milk-administered group 3 days after infection was significantly decreased compared with that in the control group. CONCLUSIONS: These results suggest that oral administration of milk fermented with L. cremoris FC protects mice against IFV infection. SIGNIFICANCE AND IMPACT OF THE STUDY: These results demonstrate that oral administration of milk fermented with exopolysaccharide-producing Lactococcus strains might protect host animals against IFV infection.

Enhanced clinical mastitis resistance in Holsteins with a FEZL p.Gly105(12_13) polymorphism.

Mastitis is a common infectious disease of the mammary gland and a major problem in the dairy industry. We previously reported that forebrain embryonic zinc finger-like (FEZL) encoding a stretch of 12 glycines (p.Gly105[12]) instead of 13 glycines (p.Gly105[13]) is associated with a lower somatic cell score (SCS) in a family derived from Walkway Chief Mark. Here we report that the p.Gly105[12] allele is associated with a significantly decreased incidence of clinical mastitis in a large Holstein population. We genotyped the FEZL polymorphism in 918 randomly collected Holstein sires, and investigated the effect of the polymorphism on the estimated breeding value (EBV) for SCS and milk, fat, solids-not-fat, and protein yield, and on the number of cattle with clinical mastitis among daughters derived from these sires. The average EBV for SCS among sires carrying the heterozygous p.Gly105[12] was significantly lower than that among sires carrying the homozygous p.Gly105[13], whereas we found no unfavorable effects of this polymorphism on EBV for milk, fat, solids-not-fat, and protein yield. The proportion of cows with clinical mastitis derived from sires carrying heterozygous p.Gly105[12] was significantly lower than that of daughters derived from sires carrying the homozygous p.Gly105[13]. Thus, selection of sires carrying p.Gly105[12] could be beneficial in the dairy industry by reducing the incidence of mastitis.

Phase-contrast X-ray imaging of the gas diffusion layer of fuel cells.

The understanding of and in situ observation of the transport and distribution of water in carbon-paper gas diffusion layers (GDLs) using non-destructive imaging techniques is critical for achieving high performance in polymer electrolyte fuel cells (PEFCs). To investigate the behavior of water in GDLs of PEFCs, phase-contrast X-ray imaging via X-ray interferometric imaging (XII) and diffraction-enhanced imaging (DEI) were performed using 35 keV X-rays. The XII technique is useful for the radiographic imaging of GDLs and in situ observations of water evolution processes in operating PEFCs. DEI provides a way for tomographic imaging of GDLs in PEFCs. Because high-energy X-rays are applicable to the imaging of both carbon papers and heavy materials, which make up PEFCs, phase-contrast X-ray imaging techniques have proven to be valuable for investigation of GDLs.

Pharmacokinetics and pharmacodynamics of basiliximab in Japanese pediatric renal transplant patients.

OBJECTIVES: This study was aimed at evaluating the pharmacokinetics and pharmacodynamics of basiliximab in Japanese pediatric renal transplant patients. MATERIALS AND METHODS: This study was carried out with the approval of the Institutional Review Board of our institution. Written consent was obtained from the legal representative of each patient, as also from the patients themselves where possible. Eligible patients were Japanese pediatric patients weighing less than 35 kg and younger than 15 years of age who were scheduled to undergo renal transplantation. Each patient was given intravenous basiliximab at the total dose of 20 mg administered in two divided doses of 10 mg each on the day of transplantation and on the fourth day after transplantation. Cyclosporine and corticosteroids were also administered as the basic concomitant maintenance immunosuppressive therapy. The time course of changes in the serum basiliximab concentrations and the percentage of CD25+ T-lymphocytes in the peripheral blood were determined up to 26 weeks after the transplantation to calculate the period of suppression of the CD25+ T-lymphocytes. Serum basiliximab was measured by an ELISA technique, and the percentage of CD25+ T-lymphocytes in the peripheral blood was determined by flow cytometry. RESULTS: 6 Japanese pediatric patients weighing less than 35 kg and aged over 1 year and less than 15 years who were scheduled to undergo renal transplantation were enrolled in this study. In regard to the time course of changes of the serum basiliximab concentration, after the peak serum concentration was reached, basiliximab was gradually eliminated from the blood with a mean half-life of 7.06 days. CD25+ T-lymphocytes in the peripheral blood were suppressed completely when the serum concentration of basiliximab was over 0.2 microg/ml, and the period of suppression of the CD25+ T cells was 40.3 - 51.7 days (mean +/- SD; 45.8 +/- 4.9). CONCLUSION: Changes in the serum concentration of basiliximab and the period of suppression of CD25+ peripheral blood T-lymphocytes in Japanese pediatric renal transplant patients were similar to those reported for non-Japanese pediatric transplant patients and Japanese adult renal transplant patients with a cyclosporine and corticosteroid regimen. This indicates that expected efficacy can be obtained in Japanese pediatric renal transplant patients using the recommended dosing regimens validated by non-Japanese studies.

Microtubule-severing activity in M phase.

The stable cytoplasmic microtubules that emanate from centrosomes in eukaryotic cells disappear at the onset of M phase and are replaced by the dynamic microtubules of the mitotic spindle. Microtubule-severing activity increases significantly under the control of maturation-promoting factor at the transition between G2 phase and M phase, and is thought to be involved in the microtubule reorganization. This review highlights three microtubule-severing factors that may be responsible for microtubule-severing activity in M phase.

MAP kinase is required for the spindle assembly checkpoint but is dispensable for the normal M phase entry and exit in Xenopus egg cell cycle extracts.

In Xenopus laevis egg cell cycle extracts that mimic early embryonic cell cycles, activation of MAP kinase and MAP kinase kinase occurs in M phase, slightly behind that of maturation promoting factor. To examine the possible role of MAP kinase in the in vitro cell cycle, we depleted the extracts of MAP kinase by using anti-Xenopus MAP kinase antibody. Like in the mock-treated extracts, the periodic activation and deactivation of MPF occurred normally in the MAP kinase-depleted extracts, suggesting that MAP kinase is dispensable for the normal M phase entry and exit in vitro. It has recently been reported that microtubule depolymerization by nocodazole treatment can block exit from mitosis in the extracts if enough sperm nuclei are present, and that the addition of MAP kinase-specific phosphatase MKP-1 overcomes this spindle assembly checkpoint, suggesting the involvement of MAP kinase in the checkpoint signal transduction. We show here that the spindle assembly checkpoint mechanism cannot operate in the MAP kinase-depleted extracts. But, adding recombinant Xenopus MAP kinase to the MAP kinase-depleted extracts restored the spindle assembly checkpoint. These results indicate unambiguously that classical MAP kinase is required for the spindle assembly checkpoint in the cell cycle extracts. In addition, we show that strong activation of MAP kinase by the addition of a constitutively active MAP kinase kinase kinase in the absence of sperm nuclei and nocodazole, induced mitotic arrest in the extracts. Therefore, activation of MAP kinase alone is sufficient for inducing the mitotic arrest in vitro.

Phenylmercuric acetate: metabolic conversion by microorganisms.

Phenylmercuric acetate, an organomercurial that has been widely used as a fungicide and slimicide, was found to be metabolized quickly by soil and aquatic microorganisms. One of the major metabolic products was identified to be diphenylmercury. In none of the cases has a methylmercury derivative been found among the microbial metabolic products of phenylmercuric acetate.

Microtubule severing by elongation factor 1 alpha.

An activity that severs stable microtubules is thought to be involved in microtubule reorganization during the cell cycle. Here, a 48-kilodalton microtubule-severing protein was purified from Xenopus eggs and identified as translational elongation factor 1 alpha (EF-1 alpha). Bacterially expressed human EF-1 alpha also displayed microtubule-severing activity in vitro and, when microinjected into fibroblasts, induced rapid and transient fragmentation of cytoplasmic microtubule arrays. Thus, EF-1 alpha, an essential component of the eukaryotic translational apparatus, appears to have a second role as a regulator of cytoskeletal rearrangements.

Crystal Structure of the High-Pressure Phase of Solid CO2.

X-ray diffraction study of solid CO(2) at room temperature has shown that the powder pattern of the high-pressure phase, which supersedes the low-pressure cubic Pa3 phase at about 10 gigapascals, is consistently interpreted in terms of an orthorhombic Cmca structure. The orthorhombic cell at 11.8 gigapascals has dimensions of 4.330 +/- 0.015, 4.657 +/- 0.005, 5.963 +/- 0.009 angstroms for its a, b, and c faces, respectively, and a volume of 120.3 +/- 0.5 cubic angstroms. Four molecules contained in the unit cell are located at the base-centered positions with their molecular axes inclined at about 52 degrees with respect to the crystallographic c axis. The volume change associated with the Pa3-Cmca transition is close to zero. The structural dimensions obtained for the high-pressure crystalline phase of CO(2) are of great importance for a theoretical understanding of the role of intermolecular interactions, including quadrupole-quadrupole interactions, in molecular condensation.

Stimulatory effects of yeast and mammalian 14-3-3 proteins on the Raf protein kinase.

Intracellular signaling from receptor tyrosine kinases in mammalian cells results in activation of a signal cascade that includes the guanine nucleotide-binding protein Ras and the protein kinases Raf, MEK [mitogen-activated protein kinase (MAPK) or extracellular signal-regulated kinase (ERK) kinase], and MAPK. MAPK activation that is dependent on the coupling of Ras and Raf was reconstituted in yeast. Yeast genes were isolated that, when overexpressed, enhanced the function of Raf. One of them is identical to BMH1, which encodes a protein similar to members of the mammalian 14-3-3 family. Bacterially synthesized mammalian 14-3-3 protein stimulated the activity of Raf prepared from yeast cells expressing c-Raf-1. Thus, the 14-3-3 protein may participate in or be required for activation of Raf.

TAB1: an activator of the TAK1 MAPKKK in TGF-beta signal transduction.

Transforming growth factor-beta (TGF-beta) regulates many aspects of cellular function. A member of the mitogen-activated protein kinase kinase kinase (MAPKKK) family, TAK1, was previously identified as a mediator in the signaling pathway of TGF-beta superfamily members. The yeast two-hybrid system has now revealed two human proteins, termed TAB1 and TAB2 (for TAK1 binding protein), that interact with TAK1. TAB1 and TAK1 were co-immunoprecipitated from mammalian cells. Overproduction of TAB1 enhanced activity of the plasminogen activator inhibitor 1 gene promoter, which is regulated by TGF-beta, and increased the kinase activity of TAK1. TAB1 may function as an activator of the TAK1 MAPKKK in TGF-beta signal transduction.

Induction of apoptosis by ASK1, a mammalian MAPKKK that activates SAPK/JNK and p38 signaling pathways.

Mitogen-activated protein (MAP) kinase cascades are activated in response to various extracellular stimuli, including growth factors and environmental stresses. A MAP kinase kinase kinase (MAPKKK), termed ASK1, was identified that activated two different subgroups of MAP kinase kinases (MAPKK), SEK1 (or MKK4) and MKK3/MAPKK6 (or MKK6), which in turn activated stress-activated protein kinase (SAPK, also known as JNK; c-Jun amino-terminal kinase) and p38 subgroups of MAP kinases, respectively. Overexpression of ASK1 induced apoptotic cell death, and ASK1 was activated in cells treated with tumor necrosis factor-alpha (TNF-alpha). Moreover, TNF-alpha-induced apoptosis was inhibited by a catalytically inactive form of ASK1. ASK1 may be a key element in the mechanism of stress- and cytokine-induced apoptosis.

Activation of the estrogen receptor through phosphorylation by mitogen-activated protein kinase.

The phosphorylation of the human estrogen receptor (ER) serine residue at position 118 is required for full activity of the ER activation function 1 (AF-1). This Ser118 is phosphorylated by mitogen-activated protein kinase (MAPK) in vitro and in cells treated with epidermal growth factor (EGF) and insulin-like growth factor (IGF) in vivo. Overexpression of MAPK kinase (MAPKK) or of the guanine nucleotide binding protein Ras, both of which activate MAPK, enhanced estrogen-induced and antiestrogen (tamoxifen)-induced transcriptional activity of wild-type ER, but not that of a mutant ER with an alanine in place of Ser118. Thus, the activity of the amino-terminal AF-1 of the ER is modulated by the phosphorylation of Ser118 through the Ras-MAPK cascade of the growth factor signaling pathways.

The MAP kinase cascade is essential for diverse signal transduction pathways.

Mitogen-activated protein (MAP) kinases are activated by combined tyrosine and threonine phosphorylation catalysed by MAP kinase kinase, a novel class of protein kinases with dual specificity for both tyrosine and serine/threonine. MAP kinase kinase is turned on by serine/threonine phosphorylation catalysed by an immediate upstream kinase. The MAP kinase cascade appears to be conserved during evolution and thus might play an essential role in diverse intracellular signaling processes from yeasts to vertebrates.

Akt mediates Rac/Cdc42-regulated cell motility in growth factor-stimulated cells and in invasive PTEN knockout cells.

Growth factors promote cell survival and cell motility, presumably through the activation of Akt and the Rac and Cdc42 GTPases, respectively. Because Akt is dispensable for Rac/Cdc42 regulation of actin reorganization, it has been assumed that Rac and Cdc42 stimulate cell motility independent of Akt in mammalian cells. However, in this study we demonstrate that Akt is essential for Rac/Cdc42-regulated cell motility in mammalian fibroblasts. A dominant-negative Akt inhibits cell motility stimulated by Rac/Cdc42 or by PDGF treatment, without affecting ruffling membrane-type actin reorganization. We have confirmed a previous report that Akt is activated by expression of Rac and Cdc42 and also observed colocalization of endogenous phosphorylated Akt with Rac and Cdc42 at the leading edge of fibroblasts. Importantly, expression of active Akt but not the closely related kinase SGK is sufficient for increasing cell motility. This effect of Akt is cell autonomous and not mediated by inhibition of GSK3. Finally, we found that dominant-negative Akt but not SGK reverses the increased cell motility phenotype of fibroblasts lacking the PTEN tumor suppressor gene. Taken together, these results suggest that Akt promotes cell motility downstream of Rac/Cdc42 in growth factor-stimulated cells and in invasive PTEN-deficient cells.

Schizosaccharomyces pombe Spk1 is a tyrosine-phosphorylated protein functionally related to Xenopus mitogen-activated protein kinase.

Mitogen-activated protein kinase (MAPK) and its direct activator, MAPK kinase (MAPKK), have been suggested to play a pivotal role in a variety of signal transduction pathways in higher eukaryotes. The fission yeast Schizosaccharomyces pombe carries a gene, named spk1, whose product is structurally related to vertebrate MAPK. Here we show that Spk1 is functionally related to Xenopus MAPK. (i) Xenopus MAPK partially complemented a defect in the spk1- mutant. An spk1- diploid strain could not sporulate, but one carrying Xenopus MAPK could. (ii) Both Spk1 and Xenopus MAPK interfered with sporulation if overexpressed in S. pombe cells. (iii) Spk1 underwent tyrosine phosphorylation as does Xenopus MAPK. Tyrosine phosphorylation of Spk1 appeared to be dependent upon mating signals because it occurred in homothallic cells but not in heterothallic cells. Furthermore, this phosphorylation was diminished in a byr1 disruptant strain, suggesting that spk1 lies downstream of byr1, which encodes a MAPKK homolog in S. pombe. Taken together, the MAPKK-MAPK cascade may be evolutionarily conserved in signaling pathways in yeasts and vertebrates.

A yeast mitogen-activated protein kinase homolog (Mpk1p) mediates signalling by protein kinase C.

Mitogen-activated protein (MAP) kinases are activated in response to a variety of stimuli through a protein kinase cascade that results in their phosphorylation on tyrosine and threonine residues. The molecular nature of this cascade is just beginning to emerge. Here we report the isolation of a Saccharomyces cerevisiae gene encoding a functional analog of mammalian MAP kinases, designated MPK1 (for MAP kinase). The MPK1 gene was isolated as a dosage-dependent suppressor of the cell lysis defect associated with deletion of the BCK1 gene. The BCK1 gene is also predicted to encode a protein kinase which has been proposed to function downstream of the protein kinase C isozyme encoded by PKC1. The MPK1 gene possesses a 1.5-kb uninterrupted open reading frame predicted to encode a 53-kDa protein. The predicted Mpk1 protein (Mpk1p) shares 48 to 50% sequence identity with Xenopus MAP kinase and with the yeast mating pheromone response pathway components, Fus3p and Kss1p. Deletion of MPK1 resulted in a temperature-dependent cell lysis defect that was virtually indistinguishable from that resulting from deletion of BCK1, suggesting that the protein kinases encoded by these genes function in a common pathway. Expression of Xenopus MAP kinase suppressed the defect associated with loss of MPK1 but not the mating-related defects associated with loss of FUS3 or KSS1, indicating functional conservation between the former two protein kinases. Mutation of the presumptive phosphorylated tyrosine and threonine residues of Mpk1p individually to phenylalanine and alanine, respectively, severely impaired Mpk1p function. Additional epistasis experiments, and the overall architectural similarity between the PKC1-mediated pathway and the pheromone response pathway, suggest that Pkc1p regulates a protein kinase cascade in which Bck1p activates a pair of protein kinases, designated Mkk1p and Mkk2p (for MAP kinase-kinase), which in turn activate Mpk1p.

The fission yeast pmk1+ gene encodes a novel mitogen-activated protein kinase homolog which regulates cell integrity and functions coordinately with the protein kinase C pathway.

We have isolated a gene, pmk1+, a third mitogen-activated protein kinase (MAPK) gene homolog from the fission yeast Schizosaccharomyces pombe. The predicted amino acid sequence shows the most homology (63 to 65% identity) to those of budding yeast Saccharomyces Mpk1 and Candida Mkc1. The Pmk1 protein contains phosphorylated tyrosines, and the level of tyrosine phosphorylation was increased in the dsp1 mutant which lacks an attenuating phosphatase for Pmk1. The level of tyrosine phosphorylation appears constant during hypotonic or heat shock treatment. The cells with pmk1 deleted (delta pmk1) are viable but show various defective phenotypes, including cell wall weakness, abnormal cell shape, a cytokinesis defect, and altered sensitivities to cations, such as hypersensitivity to potassium and resistance to sodium. Consistent with a high degree of conservation of amino acid sequence, multicopy plasmids containing the MPK1 gene rescued the defective phenotypes of the delta pmk1 mutant. The frog MAPK gene also suppressed the pmk1 disruptant. The results of genetic analysis indicated that Pmk1 lies on a novel MAPK pathway which does not overlap functionally with the other two MAPK pathways, the Spk1-dependent mating signal pathway and Sty1/Spc1/Phh1-dependent stress-sensing pathway. In Saccharomyces cerevisiae, Mpk1 is involved in cell wall integrity and functions downstream of the protein kinase C homolog. In contrast, in S. pombe, Pmk1 may not act in a linear manner with respect to fission yeast protein kinase C homologs. Interestingly, however, these two pathways are not independent; instead, they regulate cell integrity in a coordinate manner.