Liver Resident Macrophages (Kupffer Cells) Share Several Functional Antigens in Common with Endothelial Cells.

The identification and specific functions of Kupffer cells (KCs), a liver resident macrophage subpopulation, are still unclear. We compared KCs with peritoneal macrophages using cDNA microarray analysis and found that these cells share some antigens with endothelial cells. KCs highly express VCAM-1 and VEGF receptors (VEGF-Rs) at transcriptional and protein levels. VCAM-1 mediates the functional binding of KCs with lymphocytes and induces KC activation. Among the VEGF receptors, VEGF-R2 and VEGF-R3 were expressed on the KCs, while VEGF-R1 was expressed on other tissue macrophage subsets. VEGF120, a ligand of both VEGF-R1 and VEGF-R2, transduced strong survival and chemotactic signals through the KCs, when compared to PIGF, a VEGF-R1 ligand, indicating that VEGF-R2 plays significant roles in regulating KC activities. Expression of the VEGF-Rs was regulated by TLR4 signalling. These results suggest that the function of KCs is partly regulated by the common antigens shared with endothelial cells.

Fasudil, a rho kinase inhibitor, limits motor neuron loss in experimental models of amyotrophic lateral sclerosis.

BACKGROUND AND PURPOSE: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder with no effective treatment. Fasudil hydrochloride (fasudil), a potent rho kinase (ROCK) inhibitor, is useful for the treatment of ischaemic diseases. In previous reports, fasudil improved pathology in mouse models of Alzheimer's disease and spinal muscular atrophy, but there is no evidence in that it can affect ALS. We therefore investigated its effects on experimental models of ALS. EXPERIMENTAL APPROACH: In mice motor neuron (NSC34) cells, the neuroprotective effect of hydroxyfasudil (M3), an active metabolite of fasudil, and its mechanism were evaluated. Moreover, the effects of fasudil, 30 and 100 mg·kg(-1), administered via drinking water to mutant superoxide dismutase 1 (SOD1(G93A)) mice were tested by measuring motor performance, survival time and histological changes, and its mechanism investigated. KEY RESULTS: M3 prevented motor neuron cell death induced by SOD1(G93A). Furthermore, M3 suppressed both the increase in ROCK activity and phosphorylated phosphatase and tensin homologue deleted on chromosome 10 (PTEN), and the reduction in phosphorylated Akt induced by SOD1(G93A). These effects of M3 were attenuated by treatment with a PI3K inhibitor (LY294002). Moreover, fasudil slowed disease progression, increased survival time and reduced motor neuron loss, in SOD1(G93A) mice. Fasudil also attenuated the increase in ROCK activity and PTEN, and the reduction in Akt in SOD1(G93A) mice. CONCLUSIONS AND IMPLICATIONS: These findings indicate that fasudil may be effective at suppressing motor neuron degeneration and symptom progression in ALS. Hence, fasudil may have potential as a therapeutic agent for ALS treatment.

A randomized open trial for comparison of proton pump inhibitors, omeprazole versus rabeprazole, in dual therapy for Helicobacter pylori infection in relation to CYP2C19 genetic polymorphism.

BACKGROUND AND AIM: The genetic polymorphism of cytochrome P450 (CYP) 2C19 has been shown to influence the efficacy of Helicobacter pylori eradication therapy with a proton pump inhibitor (PPI) and amoxicillin (so-called dual therapy). Omeprazole, a widely used PPI, and rabeprazole, a new PPI, are metabolized in different pathways in terms of CYP2C19 genetic polymorphisms. In this study, we compared the efficacy of omeprazole and rabeprazole in a 2-week dual therapy in relation to CYP2C19 polymorphism. METHODS: One hundred and ninety-nine patients with peptic ulcer disease were randomly assigned to receive one of the following regimens: 500 mg t.i.d. amoxicillin together with either 20 mg b.i.d. omeprazole or 10 mg b.i.d rabeprazole. The eradication of H. pylori was evaluated by using a bacterial culture and a [(13)C]-urea breath test at 1--2 months after completion of treatment. Cytochrome P4502C19 polymorphism was analyzed by using polymerase chain reaction-restriction fragment length polymorphism. RESULTS: Intention-to-treat-based cure rates for the omeprazole or rabeprazole regimens were 66.3% (95% CI, 56--75) and 62.4% (95% CI, 52--71), respectively, without significant difference. Cytochrome P4502C19 genetic polymorphism did not influence the cure rates in either of these regimens. We analyzed various factors associated with treatment failure (PPI, CYP2C19 genotype, and smoking habit) by using multiple logistic regression; smoking was the only significant independent factor for treatment failure. CONCLUSION: Omeprazole and rabeprazole were equally effective in combination with amoxicillin in eradicating H. pylori, irrespective of the PPI used (omeprazole or rabeprazole) and CYP2C19 genetic polymorphism. Smoking significantly decreased the cure rate of H. pylori infection in the dual therapy.

Coordinate involvement of cell cycle arrest and apoptosis strengthen the effect of FTY720.

A novel reagent, FTY720 (2-amino-2-[2-(4-octylphenyl)ethyl]-1,3-propanediol hydrochloride), has been shown to induce a significant decrease of lymphocytes and lymphoma cells and is expected to be a potent immunosuppressant and anti-tumor drug. The decrease in lymphocytes and lymphoma cells is mainly the result of FTY720-induced apoptosis. FTY720 directly affects mitochondria and induces cell death. Moreover, FTY720 activates protein phosphatase (PP) 2A and affects anti-apoptotic intracellular signal transduction proteins to attenuate the anti-apoptotic effect. In this study, we examined the relationship between FTY720-induced apoptosis and cell cycle regulation. FTY720 induced apoptosis significantly at the G0 / G1 phase and caused G0 / G1 cell cycle arrest of the human lymphoma cell lines HL-60RG and Jurkat. Simultaneously, retinoblastoma protein (pRB) was dephosphorylated, suggesting that dephosphorylation of pRB was related to FTY720-induced G0 / G1 cell cycle arrest. Because this dephosphorylation was completely blocked by a specific PP1 / 2A inhibitor, okadaic acid, it appears that FTY720-activated PP2A is essential for FTY720-induced cell cycle arrest. FTY720-induced apoptosis was inhibited by Bcl-2 overexpression in Jurkat cells, but this did not prevent FTY720-induced cell cycle arrest, suggesting that the mechanism of FTY720-induced cell cycle arrest is independent of the mechanism of FTY720-induced apoptosis. These two independent pathways strengthen the effect of FTY720.

Immunosuppressant FTY720 induces apoptosis by direct induction of permeability transition and release of cytochrome c from mitochondria.

FTY720 has immunosuppressive activity in experimental organ transplantation and shows a prompt and protracted decrease of blood T lymphocytes upon oral administration. The blood lymphocyte decrease in vivo was mainly a result of FTY720-induced apoptosis. However, this apoptotic mechanism is not well understood. We examined the mechanism of FTY720-induced apoptosis in lymphoma. Western blotting and fluorescent caspase-specific substrate revealed that caspase-3 is involved in FTY720-induced apoptosis, whereas caspase-1 is not. Apoptotic cell death was inhibited by the pan-caspase inhibitor, Z-VAD-FMK, suggesting that caspase activation is essential for FTY720-induced apoptosis. FTY720 reduced mitochondrial transmembrane potential and released cytochrome c from the mitochondria of intact cells as well as in a cell-free system even in the presence of Z-VAD-FMK. As these mitochondrial reactions occurred before caspase activation, we concluded that FTY720 directly influences mitochondrial functions. The inhibition of mitochondrial permeability transition by Bcl-2 overexpression or by chemical inhibitors prevented all apoptotic events occurring in intact cells and in a cell-free system. Moreover, using a cell-free system, FTY720 did not directly affect isolated nuclei or cytosol. These results indicate that FTY720 directly affects mitochondria and triggers permeability transition to induce further apoptotic events.

Evidence that FTY720 induces T cell apoptosis in vivo.

The immunosuppressant FTY720 induces a drastic decrease in blood lymphocytes, especially T cells; a decrease which is assumed to be the immunosuppressive mechanism of this drug. FTY720 causes cell death in vitro in lymphocytes and leukemia cells. However, the deletion mechanism of blood lymphocytes in vivo remains unclear. We investigated whether administration of FTY720 induced lymphocyte apoptosis in blood circulation. A marked decrease in the number of blood lymphocytes was observed within an hour after a single oral administration of FTY720 at doses of 5-10 mg/kg in rats and mice. Experiments using fluorescein isothiocyanate (FITC)-Annexin V and APO-BRDU methods revealed that FTY720 induced blood lymphocyte apoptosis in a dose-dependent manner. On the other hand, lymphocyte homing to Peyer's patches was proposed as the mechanism underlying the blood lymphocyte decrease at these doses. However, similar results were obtained when using aly/aly mice, which lack Peyer's patches and lymph nodes. Thus, we concluded that apoptosis of blood lymphocytes was induced immediately after administration of FTY720, and the cells could be immediately scavenged by phagocytes or the reticuloendothelial system in addition to Peyer's patches homing. We also concluded that T cells were highly sensitive to FTY720, which induced apoptosis in vivo.

Structure of KW-2228, a tailored human granulocyte colony-stimulating factor with enhanced biological activity and stability.

KW-2228 is a tailored human granulocyte colony-stimulating factor (hG-CSF) which has more potent granulopoietic activity and is more stable than wild-type hG-CSF. Analysis of the 2.3 A resolution crystal structure of KW-2228 unambiguously revealed a four-alpha-helix bundle motif with up-up-down-down connectivity. The structures of long overhand loops connecting the helices and the N-terminus have been definitively determined. The present analysis has clearly revealed that substituted residues play important roles in fastening a long overhand loop to the N- and C-termini to fix the conformation. This conformation should be responsible for a substantial enhancement of the biological activity and stability.

(E)-4-(2-[[3-(indol-5-yl)-1-oxo-2-butenyl]amino]phenoxy)butyric acid derivatives: a new class of steroid 5 alpha-reductase inhibitors in the rat prostate. 1.

A series of (E)-4-(2-[[3-(indol-5-yl)-1-oxo-2-butenyl]amino]phenoxy)butyric acid derivatives was prepared, and the derivatives were demonstrated to be potent inhibitors of steroid 5 alpha-reductase in the rat prostate. The structure-activity relationships were as follows. An alpha-branched alkyl or benzyl substituent of proper size at position 1 of the indole is crucial for optimal enzyme inhibitory activity. N-Methylation of the amide NH resulted in complete loss of activity. Thus, coplanarity of the benzene ring and amide moiety is essential for such activity. Among the compounds prepared, (E)-4-(2-[[3-[1-[bis(4-fluorophenyl)methyl]indol-5-yl]-1-oxo-2- butenyl]-amino]phenoxy)butyric acid (57, KF18678) was one of the most potent compounds (rat prostate 5 alpha-reductase IC50 = 3.3 nM).

Improved method of preparation of inflated-fixed lung for radiologic-pathologic correlation.

The technique of inflation and fixation of the lung with polyethylene glycol is useful for specimen radiography and radiologic-pathologic correlation, but it is limited by poor histologic staining of the fixed tissue. To improve staining we used formalin to distend and fix the lung before standard fixation with a polyethylene glycol mixture. In this preliminary study, canine and infant lungs, and lungs from three cases of lung cancer were examined. The modified technique provided high-quality staining and satisfactory specimen radiography in all cases except one of the lung cancers; in this case excessive shrinkage occurred and degraded radiographic quality. We conclude that the new method of preparation permits both specimen radiography and high quality staining of the fixed tissue.

Crystallization and preliminary diffraction studies of recombinant human granulocyte-stimulating factor (KW2228).

Human granulocyte colony-stimulating factor (hG-CSF) specifically stimulates proliferation of neutrophils. Two crystal forms of a mutant of hG-CSF expressed in Escherichia coli have been obtained using the hanging drop vapour diffusion method. One form is triclinic, space group P1, with cell dimensions a = 37.3 A, b = 46.4 A, c = 47.7 A, alpha = 105.5 degrees, beta = 98.0 degrees and gamma = 109.4 degrees. The other is monoclinic, space group C2, with cell dimensions a = 82.0 A, b = 49.2 A, c = 49.4 A and beta = 113.9 degrees. Both crystal forms diffract beyond 2.0 A and are suitable for X-ray analysis.

Tertiary structure of Bacillus thermoproteolyticus [4Fe-4S] ferredoxin. Evolutionary implications for bacterial ferredoxins.

The structure of a low-potential [4Fe-4S] ferredoxin from Bacillus thermoproteolyticus has been solved using anomalous scattering data from iron atoms in the diffraction data of native crystals and refined partially to a crystallographic R-factor of 0.33, with 2.3 A (1 A = 0.1 nm) resolution data. The least-squares refinement based on the Bijvoet differences has determined that the four iron atoms in the cluster are an equal distance, approximately 2.8 A, apart. The NH ... S hydrogen bonds between polypeptide nitrogen atoms, and both cysteine and inorganic sulfur atoms, are present, as in ferrodoxin from Peptococcus aerogenes. The polypeptide chain of the B. thermoproteolyticus ferredoxin has a fold closely similar to that of 2[4Fe-4S] ferredoxin from P. aerogenes. The structural correspondence indicates strongly that both types of ferredoxin evolved from a common ancestor. The second cluster-binding region in P. aerogenes ferredoxin corresponds to the alpha-helix in B. thermoproteolyticus ferredoxin. The secondary-structure predictions strongly suggest that the alpha-helix is generally present in the monocluster-type ferredoxins. The conformational change to alpha-helix, insertions of a loop and a protrusion, as well as the absence of the second cluster in B. thermoproteolyticus ferredoxin, result in the lack of 2-fold symmetry present in P. aerogenes ferredoxin. So, the track of gene duplication is no longer detectable in the tertiary structure alone. The evolutionary events that may have occurred in the ferredoxins with the [4Fe-4S] cluster are discussed.

Crystallization and preliminary characterization of arabis mosaic virus.

Arabis mosaic virus, a member of the nepovirus group, was purified by a combination of differential centrifugation, gel filtration, and sucrose density gradient techniques. The molecular weight of the coat protein, estimated by SDS-polyacrylamide gel electrophoresis, and consideration of subunit packing indicated the surface lattice of the virion to be T = 1. The virus was stable to treatment with trypsin at neutral pH. The virus crystallizes in the cubic system, space group P2(1)3 with a = 387 A . There are four particles per unit cell, each situated on a crystallographic threefold axis.

Possible site of negative and positive feedback action of oestrogen on gonadotrophin secretion in normal women.

For determination of the site of action of oestrogen (E) during the negative and positive feedback phases of gonadotrophin secretions, studies were made on the pituitary response to a small amount of LRH and the pulsatility of gonadotrophins after E administration in normal cycling women in the mid-follicular phase. The pituitary responses to an iv bolus of 2.5 micrograms of synthetic LRH were evaluated by measuring serum LH and FSH 2 h before and 8 h after administration of 20 mg of conjugated E (Premarin). In the next cycle, the pituitary responses to a same dose of LHRH were also observed 2 h before and 56 h after E injection. The mean levels of serum LH and FSH and the peak responses to LRH were significantly (P less than 0.05) decreased 8 h after E injection, but were significantly (P less than 0.05) increased 56 h after E administration. In the third cycle, the pulsatility of gonadotrophins was evaluated by measuring serum LH and FSH every 15 min for 180 min before and 8 h and 56 h after E injection. The pulse frequencies of gonadotrophins were not significantly different before and 8 h and 56 h after E injection. The amplitudes of pulses 56 h after Premarin injection were significantly higher than those before the injection. These findings suggest that the negative and positive feedback effects of E on gonadotrophin secretion may be caused, in part, by its direct action on the pituitary response to LRH.

Site of negative feedback action of estrogen on gonadotropin secretion in normal women.

We have reported that iv administration of conjugated estrogens results in no significant change in the plasma LH-RH level during the negative feedback phase of LH, suggesting that estrogen does not suppress LH by decreasing hypothalamic LH-RH. To determine the site of estrogen action during the negative feedback phase, we studied the pituitary response to a small amount of LH-RH after estrogen administration in normal cyclic women in the mid-follicular phase. The pituitary responses to an iv bolus of 2.5 micrograms of synthetic LH-RH were evaluated by measuring serum LH and FSH 2 h before and 8 h after administration of 20 mg of conjugated estrogens (Premarin). The mean levels of serum LH and FSH were significantly (p less than 0.05) decreased 8 h after the injection. The peak responses of LH and FSH to LH-RH were also significantly (p less than 0.05) reduced after Premarin administration. These findings suggest that the negative feedback effect of estrogen on gonadotropin secretion is caused by its direct suppression on the pituitary response to LH-RH.

Hydrocortisone elicits the effect of clomiphene citrate on luteinizing hormone-releasing hormone release in vitro.

The effect of hydrocortisone on the release of luteinizing hormone (LH) and LH-releasing hormone (LRH) in response to clomiphene citrate (clomiphene) were examined in a sequential double chamber perifusion system by perifusing the mediobasal hypothalami (MBH) and/or pituitaries excised from normal female rats in dioestrus. When the MBH and the pituitary were perifused in sequence with medium containing 5 X 10(-6) M hydrocortisone, a significant release in LH (100- 150% increase, P less than 0.01-P less than 0.05) was observed 40 min after the administration of 3 X 10(-8) mol clomiphene. Clomiphene had no effect on LH release from the pituitary when perifused in series with the MBH without basal hydrocortisone infusion. Administration of clomiphene did not cause a significant increase in LH from the pituitary perifused alone, with or without medium containing hydrocortisone. The concentration of LRH in the efflux was significantly increased 40 min after clomiphene administration when MBH was perifused with medium containing hydrocortisone, whereas clomiphene had no effect when perifused with medium only. These data indicate that hydrocortisone stimulates the effect of clomiphene on LRH release from the hypothalamus, which in turn induces LH release from the pituitary.

Precise day of ovulation determined by real-time ultrasound evidence of graafian follicular development.

Real-time ultrasonic scanning was performed in 21 infertile Japanese women during 37 menstrual cycles. The maximum diameter prior to ovulation was 23.3 +/- 2.9 mm in spontaneous ovulation cycles, 29.6 +/- 5.2 mm in case of clomiphene therapies, and 26.7 +/- 3.9 mm in HMG-HCG therapies, respectively. Size of the graafian follicles was maximum at almost the same time as the LH peak in the plasma and urine, respectively. The LH peak in the urine was determined by the hemagglutination inhibition assay, the results of which were obtainable within 2 h. Four patients became pregnant (19.0%). There was no statistical correlation between the diameter of the largest follicle and the plasma estradiols (r = 0.28, 0.2 less than P less than 0.3) or between the diameter of the largest follicle and the peak luteinising hormone level (r = 0.27, 0.3 less than P less than 0.4). Therefore, the combination of the real-time ultrasound and a hemagglutination inhibition assay for LH in urine can be clinically applied to detect the precise day of the ovulation.