Upregulation of thromboxane synthase in human colorectal carcinoma and the cancer cell proliferation by thromboxane A2

Tumor growth of colorectal cancers accompanies upregulation of cyclooxygenase-2, which catalyzes a conversion step from arachidonic acid to prostaglandin H(2) (PGH(2)). Here, we compared the expression levels of thromboxane synthase (TXS), which catalyzes the conversion of PGH(2) to thromboxane A(2) (TXA(2)), between human colorectal cancer tissue and its accompanying normal mucosa. It was found that TXS protein was consistently upregulated in the cancer tissues from different patients. TXS was also highly expressed in human colonic cancer cell lines. Depletion of TXS protein by the antisense oligonucleotide inhibited proliferation of the cancer cells. This inhibition was rescued by the direct addition of a stable analogue of TXA(2). The present results suggest that overexpression of TXS and subsequent excess production of TXA(2) in the cancer cells may be involved in the tumor growth of human colorectum.     

Silymarin attenuated the amyloid β plaque burden and improved behavioral abnormalities in an Alzheimer's disease mouse model

Alzheimer's disease (AD) is characterized by progressive cognitive impairment and the formation of senile plaques. Silymarin, an extract of milk thistle, has long been used as a medicinal herb for liver diseases. Here we report marked suppression of amyloid β-protein (Aβ) fibril formation and neurotoxicity in PC12 cells after silymarin treatment in vitro. In vivo studies had indicated a significant reduction in brain Aβ deposition and improvement in behavioral abnormalities in amyloid precursor protein (APP) transgenic mice that had been preventively treated with a powdered diet containing 0.1% silymarin for 6 months. The silymarin-treated APP mice also showed less anxiety than the vehicle-treated APP mice. These behavioral changes were associated with a decline in Aβ oligomer production induced by silymarin intake. These results suggest that silymarin is a promising agent for the prevention of AD.     

Gene delivery to embryonic stem cells

Since the establishment of embryonic stem (ES) cells and the identification of tissue-specific stem cells, researchers have made great strides in the analysis of the natural biology of such stem cells for the development of therapeutic applications. Specifically, ES cells are capable of differentiating into all of the cell types that constitute the whole body. Thus, ES cell research promises new type of treatments and possible cures for a variety of debilitating diseases and injuries. The potential medical benefits obtained from stem cell technology are compelling and stem cell research sees a bright future. Control of the growth and differentiation of stem cells is a critical tool in the fields of regenerative medicine, tissue engineering, drug discovery, and toxicity testing. Toward such a goal, we present here an overview of gene delivery in ES cells, covering the following topics: significance of gene delivery in ES cells, stable versus transient gene delivery, cytotoxicity, suspension versus adherent cells, expertise, time, cost, viral vectors for gene transduction (lentiviruses, adenoviruses, and adeno-associated viruses, chemical methods for gene delivery, and mechanical or physical gene delivery methods (electroporation, nucleofection, microinjection, and nuclear transfer).     

Distribution of growth regulators in relation to the light-induced geotropic responsiveness in Zea roots

Growth regulators were measured in extracts from the upper and lower halves of 7-mm apical segments of horizontally oriented, red-light-irradiated and non-irradiated roots of Zea mays L. cv. Golden Cross Bantam 70 which exhibit a georesponse only after an exposure to light. Abscisic acid (ABA) was measured by gas-liquid chromatography, auxin (indole-3-acetic acid, IAA) by the Avena straight-growth assay, and an unidentified growth inhibitor by a Zea root-growth assay. The ratio of ABA in the upper and lower halves was 1.6 in the irradiated roots and 1.0 in the non-irradiated ones. The total amount of ABA after irradiation was increased by a factor of ca. 1.8. The ratio of IAA in the upper and lower halves of irradiated and non-irradiated roots was 1:3.4 and 1:2.9, respectively. The content (or activity) of an unidentified growth inhibitor was highest in the lower halves of horizontally oriented roots which had been irradiated with red light. The unidentified growth inhibitor, rather than IAA or ABA, may be the major factor in the light-induced geotropic responsiveness in Zea roots.     

Stable expression of gastric proton pump activity at the cell surface

Stable cell lines expressing the gastric proton pump alpha- and/or beta-subunits were constructed. The cell line co-expressing the alpha- and beta-subunits showed inward Rb(+) transport, which was activated by Rb(+) in a concentration-dependent manner. In the alpha+beta-expressing cell line, rapid recovery of intracellular pH was also observed after acid load, indicating that this cell line transported protons outward. These ion transport activities were inhibited by a proton pump inhibitor, 2-methyl-8-(phenylmethoxy)imidazo[1,2-a]pyridine-3-acetonitrile (SCH 28080). In a membrane fraction of the alpha+beta-expressing cell line, K(+)-stimulated ATPase (K(+)-ATPase) activity and the acylphosphorylation of the alpha-subunit were observed, both of which were also inhibited by SCH 28080. The specific activity and properties of the K(+)-ATPase were comparable to those found in the native gastric proton pump. In the stable cell lines, the alpha-subunit was retained in the intracellular compartment and was unstable in the absence of the beta-subunit, but it was stabilized and reached the cell surface in the presence of the beta-subunit. On the other hand, the beta-subunit was stable and able to travel to the cell surface in the absence of the alpha-subunit. These cell lines are ideal for the structure-function study of ion transport by the gastric proton pump as well as for characterization of the cellular regulation of surface expression of the functional proton pump.     

Development of simultaneous gas chromatography-mass spectrometric and liquid chromatography-electrospray ionization mass spectrometric determination method for the new designer drugs, N-benzylpiperazine (BZP), 1-(3-trifluoromethylphenyl)piperazine (TFMPP) and their main metabolites in urine

To prove the intake of recently controlled designer drugs, N-benzylpiperazine (BZP) and 1-(3-trifluoromethylphenyl)piperazine (TFMPP), a simple, sensitive and reliable method which allows us to simultaneously detect BZP, TFMPP and their major metabolite in human urine has been established by coupling gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS). GC-MS accompanied by trifluoroacetyl (TFA) derivatization and LC-MS analyses were performed after the enzymatic hydrolysis and the solid phase extraction with OASIS HLB, and BZP, TFMPP and their major metabolites, 4'-hydroxy-BZP (p-OH-BZP), 3'-hydroxy-BZP (m-OH-BZP) and 4'-hydroxy-TFMPP (p-OH-TFMPP), have found to be satisfactorily separated on a semi-micro SCX column with acetonitrile-40 mM ammonium acetate buffer (pH 4) (75:25, v/v) as the eluent. The detection limits produced by GC-MS were estimated to be from 50 ng/ml to 1 microg/ml in the scan mode, and from 200 to 500 ng/ml in the selected ion monitoring (SIM) mode. Upon applying the LC-ESI-MS technique, the linear calibration curves were obtained by using the SIM mode for all analytes in the concentration range from 10 ng/ml to 10 microg/ml. The detection limits ranged from 5 to 40 ng/ml in the scan mode, and from 0.2 to 1 ng/ml in the SIM mode. These results indicate the high reliability and sensitivity of the present procedure, and this procedure will be applicable for proof of intake of BZP and TFMPP in forensic toxicology.     

Glycomics of a novel type-2 N-acetyllactosamine-specific lectin purified from the feather star, Oxycomanthus japonicus (Pelmatozoa: Crinoidea)

A lectin - designated OXYL for the purposes of this study that strongly recognizes complex-type oligosaccharides of serum glycoproteins - was purified from a crinoid, the feather star Oxycomanthus japonicus, the most basal group among extant echinoderms. OXYL was purified through a combination of anion-exchange and affinity chromatography using Q-sepharose and fetuin-sepharose gel, respectively. Lectin was determined to be a 14-kDa polypeptide by sodium dodecyl sulphate-polyacrylamide gel electrophoresis under reducing conditions. However, 14-kDa and 28-kDa bands appeared in the same proportion under non-reducing conditions. Gel permeation chromatography showed a 54-kDa peak, suggesting that lectin consists of four 14-kDa subunits. Divalent cations were not indicated, and stable haemagglutination activity was demonstrated at pH 4-12 and temperatures below 60°C. Surface plasmon resonance analysis of OXYL against fetuin showed k(ass) and k(diss) values of 1.4×10(-6)M(-1)s(-1) and 3.1×10(-3)s(-1), respectively, indicating that it has a strong binding affinity to the glycoprotein as lectin. Frontal affinity chromatography using 25 types of prydylamine-conjugated glycans indicated that OXYL specifically recognizes multi-antennary complex-type oligosaccharides containing type-2 N-acetyllactosamines (Galβ1-4GlcNAc) if α2-3-linked sialic acid is linked at the non-reducing terminal. However, type-1 N-acetyllactosamine (Galβ1-3GlcNAc) chains and α2-6-linked sialic acids were never recognized by OXYL. This profiling study showed that OXYL essentially recognizes β1-4-linkage at C-1 position and free OH group at C-6 position of Gal in addition to the conservation of N-acetyl groups at C-2 position and free OH groups at C-3 position of GlcNAc in N-acetyllactosamine. This is the first report on glycomics on a lectin purified from an echinoderm belonging to the subphylum Pelmatozoa.     

Transmission ratio distortion of molecular markers in a doubled haploid population originated from a natural hybrid between Osmunda japonica and O. lancea

In ferns, intra-gametophytic selfing occurs as a mode of reproduction where two gametes from the same gametophyte form a completely homozygous sporophyte. Intra-gametophytic selfing is considered to be prevented by lethal or deleterious recessive genes in several diploid species. In order to investigate the modes and tempo of selection acting different developmental stages, doubled haploids obtained from intra-gametophytic selfing within isolated gametophytes of a putative F1 hybrid between Osmunda japonica and O. lancea were analyzed with EST_derived molecular markers, and the distribution pattern of transmission ratio distortion (TRD) along linkage map was clarified. As the results, the markers with skewness were clustered in two linkage groups. For the two highly distorted regions, gametophytes and F2 population were also examined. The markers skewed towards O. japonica on a linkage group (LG_2) showed skewness also in gametophytes, and the TRD was generated in the process of spore formation or growth of gametophytes. Also, selection appeared to be operating in the gametophytic stage. The markers on other linkage group (LG_11) showed highest skewness towards O. lancea in doubled haploids, and it was suggested that the segregation of LG_11 were influenced by zygotic lethality or genotypic evaluation and that some deleterious recessive genes exist in LG_11 and reduce the viability of homozygotes with O. japonica alleles. It is very likely that a region of LG_11were responsible for the low frequencies of intra-gametophytic selfing in O. japonica.     

Single Quantum Dot Tracking Reveals that an Individual Multivalent HIV-1 Tat Protein Transduction Domain Can Activate Machinery for Lateral Transport and Endocytosis

The mechanisms underlying the cellular entry of the HIV-1 Tat protein transduction domain (TatP) and the molecular information necessary to improve the transduction efficiency of TatP remain unclear due to the technical limitations for direct visualization of TatP''s behavior in cells. Using confocal microscopy, total internal reflection fluorescence microscopy, and four-dimensional microscopy, we developed a single-molecule tracking assay for TatP labeled with quantum dots (QDs) to examine the kinetics of TatP initially and immediately before, at the beginning of, and immediately after entry into living cells. We report that even when the number of multivalent TatP (mTatP)-QDs bound to a cell was low, each single mTatP-QD first locally induced the cell''s lateral transport machinery to move the mTatP-QD toward the center of the cell body upon cross-linking of heparan sulfate proteoglycans. The centripetal and lateral movements were linked to the integrity and flow of actomyosin and microtubules. Individual mTatP underwent lipid raft-mediated temporal confinement, followed by complete immobilization, which ultimately led to endocytotic internalization. However, bivalent TatP did not sufficiently promote either cell surface movement or internalization. Together, these findings provide clues regarding the mechanisms of TatP cell entry and indicate that increasing the valence of TatP on nanoparticles allows them to behave as cargo delivery nanomachines.