Highly potent PDE4 inhibitors with therapeutic potential

The hypothesis that the dose-limiting side effects of PDE4 inhibitors could be mediated via the central nervous system prompted us to design and synthesize a hydrophilic piperidine analog to improve the side effect profile of Ariflo 1, which is an orally active second-generation PDE4 inhibitor. During evaluation of various water-soluble piperidine analogs, 2a-b, 11b-14b, and 17a showed therapeutic potential in cross-species comparison studies. The following three findings were obtained: (1) The hydroxamic acid group, a well known metal chelator, caused a marked increase of inhibitory activity. (2) Water-soluble piperidine analogs lacked the configurational isomerism of Ariflo 1 without loss of inhibitory activity. (3) Replacement of the 4-methoxy residue with a difluoromethoxy residue led to an increase of in vivo potency. Structure-activity relationships are presented. Single-dose rat pharmacokinetic data for 11b, 12b, and 17a are also presented.     

Effects of fasting on thermoregulatory processes and the daily oscillations in rats

To investigate the mechanism involved in the reduction of body core temperature (T(core)) during fasting in rats, which is selective in the light phase, we measured T(core), surface temperature, and oxygen consumption rate in fed control animals and in fasted animals on day 3 of fasting and day 4 of recovery at an ambient temperature (T(a)) of 23 degrees C by biotelemetry, infrared thermography, and indirect calorimetry, respectively. On the fasting day, 1) T(core) in the light phase decreased (P < 0.05) from the control; however, T(core) in the dark phase was unchanged, 2) tail temperature fell from the control (P < 0.05, from 30.7 +/- 0.1 to 23.9 +/- 0.1 degrees C in the dark phase and from 29.4 +/- 0.1 to 25.2 +/- 0.2 degrees C in the light phase), 3) oxygen consumption rate decreased from the control (P < 0.05, from 24.37 +/- 1.06 to 16.24 +/- 0.69 ml. min(-1). kg body wt(-0.75) in the dark phase and from 18.91 +/- 0.64 to 14.00 +/- 0.41 ml. min(-1). kg body wt(-0.75) in the light phase). All these values returned to the control levels on the recovery day. The results suggest that, in the fasting condition, T(core) in the dark phase was maintained by suppression of the heat loss mechanism, despite the reduction of metabolic heat production. In contrast, the response was weakened in the light phase, decreasing T(core) greatly. Moreover, the change in the regulation of tail blood flow was a likely mechanism to suppress heat loss.     

Changes in oligosaccharide expression on plasma membrane of the mouse oocyte during fertilisation and early cleavage

It has been reported that various structural and functional changes occur on the surface of the plasma membrane of the ovum and embryo during fertilisation and cleavage in preparation for implantation. Glycoproteins are thought to be one of the factors in cell attachment. Thus, we investigated the changes in glycoprotein expression on the cell surface membrane of the mouse embryo by using lectins. Among seven types of lectin (ConA, WGA, UEA-I, MPA, LCA, DBA and PNA), the fluorescent intensities of ConA and WGA markedly increased from unfertilised ova to blastocysts. By quantitative analysis using immuno-scanning electron microscopy, the numbers of ConA-gold particles were small until 4-cell cleavage, but increased significantly at the blastocyst stage. In contrast, an increased number of WGA-gold particles was detected even at the 4-cell stage, and this increase continued to the blastocyst stage. From the above observations, we conclude that the numbers of sugar chains bound to both ConA andWGA increases with blastocyst formation and earlier expression is observed with WGA. The present study dearly shows that glycoproteins on the cell membrane surface of the mouse embryo quantitatively increase at the time of implantation, and the possibility has been indicated that glycoproteins are involved in intercellular recognition and adhesion between the embryo and endometrial epithelium.     

Structure-activity study of L-amino acid-based N-type calcium channel blockers

Synthesis and structure-activity relationship (SAR) study of L-amino acid-based N-type calcium channel blockers are described. The compounds synthesized were evaluated for inhibitory activity against both N-type and L-type calcium channels focusing on selectivity to reduce cardiovascular side effects due to blocking of L-type calcium channels. In the course of screening of our compound library, N-(t-butoxycarbonyl)-L-aspartic acid derivative 1a was identified as an initial lead compound for a new series of N-type calcium channel blockers, which inhibited calcium influx into IMR-32 human neuroblastoma cells with an IC(50) of 3.4 microM. Compound 1a also exhibited blockade of N-type calcium channel current in electrophysiological experiment using IMR-32 cells (34% inhibition at 10 microM, n=3). As a consequence of conversion of amino acid residue of 1a, compound 12a, that include N-(t-butoxycarbonyl)-L-cysteine, was found to be a potent N-type calcium channel blocker with an IC(50) of 0.61 microM. Thus, L-cysteine was selected as a potential structural motif for further modification. Optimization of C- and N-terminals of L-cysteine using S-cyclohexylmethyl-L-cysteine as a central scaffold led to potent and selective N-type calcium channel blocker 21f, which showed improved inhibitory potency (IC(50) 0.12 microM) and 12-fold selectivity for N-type calcium channels over L-type channels.     

Proteasome activator PA28gamma-dependent nuclear retention and degradation of hepatitis C virus core protein

Hepatitis C virus (HCV) core protein plays an important role in the formation of the viral nucleocapsid and a regulatory protein involved in hepatocarcinogenesis. In this study, we have identified proteasome activator PA28gamma (11S regulator gamma) as an HCV core binding protein by using yeast two-hybrid system. This interaction was demonstrated not only in cell culture but also in the livers of HCV core transgenic mice. These findings are extended to human HCV infection by the observation of this interaction in liver specimens from a patient with chronic HCV infection. Neither the interaction of HCV core protein with other PA28 subtypes nor that of PA28gamma with other Flavivirus core proteins was detected. Deletion of the PA28gamma-binding region from the HCV core protein or knockout of the PA28gamma gene led to the export of the HCV core protein from the nucleus to the cytoplasm. Overexpression of PA28gamma enhanced the proteolysis of the HCV core protein. Thus, the nuclear retention and stability of the HCV core protein is regulated via a PA28gamma-dependent pathway through which HCV pathogenesis may be exerted.     

Cytopathologic and clinicopathologic features of ovarian hepatoid carcinoma. A case report

BACKGROUND: Hepatoid carcinoma is a rare ovarian tumor and is thought to be a different histopathologic subtype from hepatoid-type yolk sac tumor based upon its pathologic features. However, the cytopathologic characteristics of ovarian hepatoid carcinoma (OHC) have not been reported previously. We report the clinicopathologic and cytopathologic features and immunoreactivity of a case of OHC. CASE: A 36-year-old woman presented to our department with lower abdominal pain. A left ovarian tumor was found on pelvic examination, magnetic resonance imaging and computed tomography. The tumor was diagnosed as a hepatoid carcinoma of the left ovary based upon the histopathology of the surgically resected specimen. Cytopathologic specimens from a tumor touch preparation of the tumor exhibited pleomorphic tumor cells with abundant cytoplasm. The nuclei contained rough, granular chromatin and large, prominent nucleoli. Several tumor cells were multinucleated. Tumor cells were immunoreactive for alpha-fetoprotein (AFP). Hematoxylin and eosin staining revealed that the tumor cells were in a sinusoidal pattern resembling hepatocellular carcinoma without any glandular formation. The tumor cells were negative for human chorionic gonadotropin while positive for AFP, alpha-1-antitripsin, CA-125 and carcinoembryonic antigen. CONCLUSION: Cytopathologic examination is of considerable aid in the diagnosis of OHC since cytopathologic preparations highlight the characteristic cell pleomorphism.     

Lipid membrane with low proton permeability

This work reports the production of a liposomal formulation, having a lipidic membrane with known chemical composition and a low proton permeability, as confirmed by physicochemical characterization of the maintenance of a transmembranic pH gradient. These liposomes consist of DSPC, DSPE-PEG, DSPG and cholesterol, with low internal pH. To verify the low proton permeability of these liposomal bilayers, a study of proton migration according to the fluorescence quenching of 9-aminoacridine (9AA), as well as CPT-11 encapsulation, were used to monitor the acidification of the intravesicular space. Both experiments showed that this liposomal formulation is able to maintain a transmembranic proton gradient.     

Larval behavioral,morphological changes,and nematocyte dynamics during settlement of actinulae of Tubularia mesembryanthemum,Allman 1871 (Hydrozoa: Tubulariidae)

The marine colonial hydroid Tubularia mesembryanthemum produces a morphologically unique dispersive stage, the actinula larva. Detailed observations were made on the behaviors and nematocyte dynamics of actinula larvae during attachment and morphogenesis by employing microscopic and time lapse video techniques. These observations produced four primary results. (1) Actinula larvae demonstrated two forms of attachment: temporary attachment by atrichous isorhiza (AI)-nematocysts discharged from the aboral tentacle (AT) tips-and permanent settlement by cement secretion from the columnar gland cells of the basal protrusion. (2) During larval settlement, numerous AIs were discharged from the AT tips with sinuous movement and rubbing of the tentacles onto the substrata, leading to "nematocyte-printing" around the settlement site. (3) Simultaneous with the discharge of the AIs, migration of stenoteles, desmonemes, and microbasic mastigophores occurred, resulting in a dramatic change of nematocyte composition in the ATs after larval settlement. This was in parallel with changes in larval behavior and the tentacle function. (4) Nematocyte-printing behavior during settlement could be recognized as metamorphic behavior responsible for irreversible changes in AT function, from attachment to feeding and defense.     

Ontogenic emergence and localization of larval skin antigen molecule recognized by adult T cells of Xenopus laevis: Regulation by thyroid hormone during metamorphosis

Results from previous studies using an inbred strain of Xenopus laevis have led to the proposition that metamorphosis includes the events by which the newly differentiating adult immune system, including T lymphocytes, recognizes and eliminates larval skin cells as 'non-self'. More recently, a larval antigen targeted by adult T cells was identified as a 59 kDa protein with a specific peptide sequence. Using antisera directed against the larval antigen and the peptide, immunohistochemistry and western blotting were done to examine expression of the 59 kDa larval antigen in the skin during larval and metamorphic periods. There was no expression before Nieuwkoop and Faber stage 53. Expression was first seen at the beginning of metamorphic stage 54, when hind limbs appear, and increased thereafter, in apical and skein cells of both trunk and tail regions. In the trunk region, expression started to decrease at stage 58, until it completely disappeared at stage 62 (metamorphic climax). In the tail skin, however, expression persisted throughout the metamorphic stages. Treatment of larvae with thyroid hormone (TH) resulted in repression of expression of the 59 kDa molecule in a dose-dependent manner. Downregulation occurred earlier in the trunk than in the tail skin. These results suggest involvement in metamorphic events of an immunological mechanism: differential expression of the larval antigen in the trunk and tail skin cells due to their differing concentration of TH results in the tail, but not the trunk skin, being selectively attacked by the newly differentiating adult-type immune system.     

Review: prediction of in vivo fates of proteins in the era of genomics and proteomics

Even after a nascent protein emerges from the ribosome, its fate is still controlled by its own amino acid sequence information. Namely, it may be co-/posttranslationally modified (e.g., phosphorylated, N-/O-glycosylated, and lipidated); it may be inserted into the membrane, translocated to an organelle, or secreted to the outside milieu; it may be processed for maturation or selective degradation; finally, its fragment may be presented on the cell surface as an antigen. Here, prediction methods of such protein fates from their amino acid sequences are reviewed. In many cases, artificial neural network techniques have been effectively used. The prediction of in vivo fates of proteins will be useful for characterizing newly identified candidate genes in a genome or for interpreting multiple spots in proteome analyses.