Goat Anti-mouse IgG Antibody Research Articles

βPak-interacting Exchange Factor-mediated Rac1 Activation Requires smgGDS Guanine Nucleotide Exchange Factor in Basic Fibroblast Growth Factor-induced Neurite Outgrowth

Neuritogenesis requires active actin cytoskeleton rearrangement in which Rho GTPases play a pivotal role. In a previous study (Shin, E. Y., Woo, K. N., Lee, C. S., Koo, S. H., Kim, Y. G., Kim, W. J., Bae, C. D., Chang, S. I., and Kim, E. G. (2004) J. Biol. Chem. 279, 1994-2004), we demonstrated that betaPak-interacting exchange factor (betaPIX) guanine nucleotide exchange factor (GEF) mediates basic fibroblast growth factor (bFGF)-stimulated Rac1 activation through phosphorylation of Ser-525 and Thr-526 at the GIT-binding domain (GBD). However, the mechanism by which this phosphorylation event regulates the Rac1-GEF activity remained elusive. We show here that betaPIX binds to Rac1 via the GBD and also activates the GTPase via an associated GEF, smgGDS, in a phosphorylation-dependent manner. Notably, the Rac1-GEF activity of betaPIX persisted for an extended period of time following bFGF stimulation, unlike other Rho GEFs containing the Dbl homology domain. We demonstrate that C-PIX, containing proline-rich, GBD, and leucine zipper domains can interact with Rac1 via the GBD in vitro and in vivo and also mediated bFGF-stimulated Rac1 activation, as determined by a modified GEF assay and fluorescence resonance energy transfer analysis. However, nonphosphorylatable C-PIX (S525A/T526A) failed to generate Rac1-GTP. Finally, betaPIX is shown to form a trimeric complex with smgGDS and Rac1; down-regulation of smgGDS expression by short interfering RNA causing significant inhibition of betaPIX-mediated Rac1 activation and neurite outgrowth. These results provide evidence for a new and unexpected mechanism whereby betaPIX can regulate Rac1 activity.

Defective Human Leukocyte Antigen Class I-associated Antigen Presentation Caused by a Novel β2-Microglobulin Loss-of-function in Melanoma Cells

The major histocompatibility complex class I molecules consist of three subunits, the 45-kDa heavy chain, the 12-kDa beta(2)-microglobulin (beta(2)m), and an approximately 8-9-residue antigenic peptide. Without beta(2)m, the major histocompatibility complex class I molecules cannot assemble, thereby abolishing their transport to the cell membrane and the subsequent recognition by antigen-specific T cells. Here we report a case of defective antigen presentation caused by the expression of a beta(2)m with a Cys-to-Trp substitution at position 25 (beta(2)m(C25W)). This substitution causes misfolding and degradation of beta(2)m(C25W) but does not result in complete lack of human leukocyte antigen (HLA) class I molecule expression on the surface of melanoma VMM5B cells. Despite HLA class I expression, VMM5B cells are not recognized by HLA class I-restricted, melanoma antigen-specific cytotoxic T lymphocytes even following loading with exogenous peptides or transduction with melanoma antigen-expressing viruses. Lysis of VMM5B cells is restored only following reconstitution with exogenous or endogenous wild-type beta(2)m protein. Together, our results indicate impairment of antigenic peptide presentation because of a dysfunctional beta(2)m and provide a mechanism for the lack of close association between HLA class I expression and susceptibility of tumor cells to cytotoxic T lymphocytes-mediated lysis in malignant diseases.

The Estrogen-responsive B Box Protein Is a Novel Regulator of the Retinoid Signal

Retinoic acid (RA) induces growth arrest, cell death, and differentiation in many human cancer cells in vitro and has entered routine clinical use for the treatment of several human cancer types. One mechanism by which cancer cells evade retinoid-induced effects is through repression of retinoic acid receptor beta (RARbeta) gene transcription. The RA response element beta (betaRARE) is the essential DNA sequence required for retinoid-induced RARbeta transcription. Here we show that the estrogen-responsive B box protein (EBBP), a member of the RING-B box-coiled-coil protein family, is a betaRARE-binding protein. EBBP undergoes serine threonine phosphorylation and enhanced protein stability after RA treatment. Following RA treatment, we also observed increased nuclear EBBP levels in aggregates with the promyelocytic leukemia protein at promyelocytic leukemia nuclear bodies. EBBP enhanced RA-responsive RARbeta transcription in RA-sensitive and -resistant cancer cells, which were resistant to both a histone deacetylase inhibitor and a demethylating agent. EBBP-specific small interfering RNA reduced basal and RA-induced RARbeta expression. EBBP increased betaRARE-transactivating function through its coiled-coil domain. Taken together, our work suggests that EBBP may have a pivotal role in the retinoid anti-cancer signal.

The Crystal Structure of H-2Db Complexed with a Partial Peptide Epitope Suggests a Major Histocompatibility Complex Class I Assembly Intermediate

In the absence of bound peptide ligands, major histocompatibility complex (MHC) class I molecules are unstable. In an attempt to determine the minimum requirement for peptide-dependent MHC class I stabilization, we have used short synthetic peptides derived from the Sendai virus nucleoprotein epitope (residues 324-332, 1FAPGNYPAL9) to promote its folding in vitro of H-2D(b). We found that H-2D(b) can be stabilized by the pentapeptide 5NYPAL9, which is equivalent to the C-terminal portion of the optimal nonapeptide and includes both the P5 and P9 anchor residues. We have crystallized the complex of the H-2D(b) molecule with the pentamer and determined the structure to show how a quasi-stable MHC class I molecule can be formed by occupancy of a single binding pocket in the peptide-binding groove.

Fibroblasts Show More Potential as Target Cells than Keratinocytes in COL7A1 Gene Therapy of Dystrophic Epidermolysis Bullosa

Dystrophic epidermolysis bullosa (DEB) is an inherited blistering skin disorder caused by mutations in the type VII collagen gene (COL7A1). Therapeutic introduction of COL7A1 into skin cells holds significant promise for the treatment of DEB. The purpose of this study was to establish an efficient retroviral transfer method for COL7A1 into DEB epidermal keratinocytes and dermal fibroblasts, and to determine which gene-transferred cells can most efficiently express collagen VII in the skin. We demonstrated that gene transfer using a combination of G protein of vesicular stomatitis virus-pseudotyped retroviral vector and retronectin introduced COL7A1 into keratinocytes and fibroblasts from a DEB patient with the lack of COL7A1 expression. Real-time polymerase chain reaction analysis of the normal human skin demonstrated that the quantity of COL7A1 expression in the epidermis was significantly higher than that in the dermis. Subsequently, we have produced skin grafts with the gene-transferred or untreated DEB keratinocytes and fibroblasts, and have transplanted them into nude rats. Interestingly, the series of skin graft experiments showed that the gene-transferred fibroblasts supplied higher amount of collagen VII to the new dermal-epidermal junction than the gene-transferred keratinocytes. An ultrastructural study revealed that collagen VII from gene-transferred cells formed proper anchoring fibrils. These results suggest that fibroblasts may be a better gene therapy target of DEB treatment than keratinocytes.

Protein Kinase C Negatively Regulates Akt Activity and Modifies UVC-induced Apoptosis in Mouse Keratinocytes

Skin keratinocytes are subject to frequent chemical and physical injury and have developed elaborate cell survival mechanisms to compensate. Among these, the Akt/protein kinase B (PKB) pathway protects keratinocytes from the toxic effects of ultraviolet light (UV). In contrast, the protein kinase C (PKC) family is involved in several keratinocyte death pathways. During an examination of potential interactions among these two pathways, we found that the insulin-like growth factor (IGF-1) activates both the PKC and the Akt signaling pathways in cultured primary mouse keratinocytes as indicated by increased phospho-PKC and phospho-Ser-473-Akt. IGF-1 also selectively induced translocation of PKCdelta and PKCepsilon from soluble to particulate fractions in mouse keratinocytes. Furthermore, the PKC-specific inhibitor, GF109203X, increased IGF-1-induced phospho-Ser-473-Akt and Akt kinase activity and enhanced IGF-1 protection from UVC-induced apoptosis. Selective activation of PKC by 12-O-tetradecanoylphorbol-13-acetate (TPA) reduced phospho-Ser-473-Akt, suggesting that activation of PKC inhibits Akt activity. TPA also attenuated IGF-1 and epidermal growth factor-induced phospho-Ser-473-Akt, reduced Akt kinase activity, and blocked IGF-1 protection from UVC-induced apoptosis. The inhibition of Akt activity by TPA was reduced by inhibitors of protein phosphatase 2A, and TPA stimulated the association of phosphatase 2A with Akt. Individual PKC isoforms were overexpressed in cultured keratinocytes by transduction with adenoviral vectors or inhibited with PKC-selective inhibitors. These studies indicated that PKCdelta and PKCepsilon were selectively potent at causing dephosphorylation of Akt and modifying cell survival, whereas PKCalpha enhanced phosphorylation of Akt on Ser-473. Our results suggested that activation of PKCdelta and PKCepsilon provide a negative regulation for Akt phosphorylation and kinase activity in mouse keratinocytes and serve as modulators of cell survival pathways in response to external stimuli.

159 LOCALIZATION OF CLAUDIN FAMILY PROTEINS IN PIG EMBRYOS DURING PRE-IMPLANTATION DEVELOPMENT

Tight junctions (TJ) are critical for blastocoel formation in mammalian pre-implantation embryos. Claudin family proteins, which are TJ proteins, are generally important for the barrier function of TJ. However, the expression and localization of claudin proteins are not clarified in mammalian pre-implantation embryos. The present study was designed to examine changes in the localization of claudin isoforms, claudin-1, -2, and -4, in pig parthenogenetic diploids through pre-implantation development. Oocyte–cumulus–granulosa cell complexes were collected from the follicles 4–6 mm in diameter, and then maturation-cultured. Only oocytes with a prominent first polar body were subjected to electro-stimulation after maturation, and they were treated with cytochalasin B to produce parthenogenetic diploids. Presumptive diploids were then cultured for 168 h and observed every 24 h. Embryos of each developmental stage from 2-cell to blastocyst were subjected to immunofluorescence staining of the anti-TJ proteins. Zona-free embryos were fixed and treated with rabbit anti-claudin-1 and -2 polyclonal antibodies or mouse anti-claudin-4 monoclonal antibody, followed by treatment with Alexa fluor 488-labeled goat anti-rabbit IgG antibody or goat anti-mouse IgG antibody. Some embryos were treated with rabbit anti-occludin antibody or anti-ZO-1 antibody for the detection of the TJ-net work. All embryos were counterstained with Hoechst 33342 and observed under an epifluorescence microscope after whole-mounting. The specific fluorescence for all TJ proteins examined was observed in embryos at all pre-implantation stages. Occludin and ZO-1 were detected in the cytoplasm before the morula stage (96 h), and localized toward the boundary region among cells from the morula and early blastocyst stages, reflecting the distribution of the tight junction in embryos. Claudin-1 and -2 localized in the cytoplasm at early-cleaving stages. They were detected in the nucleus after compaction, and the distribution of these proteins in the nucleus was dominant at the blastocyst stage. The distribution of the 2 claudin proteins in the nucleus was quite different; claudin-1 distributed rather homogeneously, but claudin-2 formed several bright spots in the nucleus. Claudin-4 also showed a unique distribution pattern in embryos; it was detected in the cytoplasm with strong fluorescence at the periphery of the nucleus of the 2- and 4-cell embryos. Claudin-4 changed its localization toward the boundary region of cells around the early blastocyst stage (120 h), and then the distribution of claudin-4 was restricted to the boundary region later in the blastocyst stage (144 h). These results indicate that claudin-4, but not claudin-1 and -2, is responsible for the formation of TJ in the pre-implantation embryos, although the function of these claudin proteins in the nucleus is unknown.

A simple and rapid immunochromatographic test strip for detection of white spot syndrome virus (WSSV) of shrimp

A simple strip-test kit for white spot syndrome virus (WSSV) detection was developed using monoclonal antibody W29 (against the VP28 structural protein) conjugated with colloidal gold as the detector antibody. A rabbit anti-recombinant VP28F118 (rVP28) protein antibody in combination with a W28 monoclonal antibody was used as the capture complex at the test line (T), and goat anti-mouse IgG antibody (GAM) was used as the capture antibody at the control line (C). For evidence, the ready-to-use strip was kept in a plastic case and stored in a desiccated plastic bag. A sample volume of 100 microl gill homogenate in application buffer was applied to the sample chamber at one end of the strip and allowed to flow by chromatography through the nitrocellulose membrane to the other end. In test samples containing WSSV, the virus bound to the monoclonal antibody conjugated with colloidal gold and the resulting complex was captured by the antibodies at T to give a reddish-purple band. Any unbound monoclonal antibody conjugated with colloidal gold moved across T to be captured by the GAM and formed a band at C. In samples without WSSV or with WSSV below the limit of detection of the kit, only the band at C was seen. This method was 4 times less sensitive than dot blotting, and about 2 000 000 times less sensitive than 1-step PCR. Nonetheless, it could be used to screen individual shrimp or pooled shrimp samples to confirm high levels of WSSV infection or WSSV disease outbreaks. The beneficial features of this kit are that simple, convenient and quick results can be obtained without the requirement of sophisticated tools or special skills.

Phosphorylation-independent Regulation of Metabotropic Glutamate Receptor 1 Signaling Requires G Protein-coupled Receptor Kinase 2 Binding to the Second Intracellular Loop

Metabotropic glutamate receptors (mGluRs) are members of a unique class of G protein-coupled receptors (class III) that include the calcium-sensing and gamma-aminobutyric acid type B receptors. The activity of mGluRs is regulated by second messenger-dependent protein kinases and G protein-coupled receptor kinases (GRKs). The attenuation of both mGluR1a and mGluR1b signaling by GRK2 is phosphorylation- and beta-arrestin-independent and requires the concomitant association of GRK2 with both the receptor and Galpha(q/11). G protein interactions are mediated, in part, by the mGluR1 intracellular second loop, but the domains required for GRK2 binding are unknown. In the present study, we showed that GRK2 binds to the second intracellular loop of mGluR1a and mGluR1b and also to the mGluR1a carboxyl-terminal tail. Alanine scanning mutagenesis revealed a discrete domain within loop 2 that contributes to GRK2 binding, and the mutation of either lysine 691 or 692 to an alanine within this domain resulted in a loss of GRK2 binding to both mGluR1a and mGluR1b. Mutation of either Lys(691) or Lys(692) prevented GRK2-mediated attenuation of mGluR1b signaling, whereas the mutation of only Lys(692) prevented GRK2-mediated inhibition of mGluR1a signaling. Thus, the mGluR1a carboxyl-terminal tail may also be involved in regulating the signaling of the mGluR1a splice variant. Taken together, our findings indicated that kinase binding to an mGluR1 domain involved in G protein-coupling is essential for the phosphorylation-independent attenuation of signaling by GRK2.

Aggravation of Anti-Myeloperoxidase Antibody-Induced Glomerulonephritis by Bacterial Lipopolysaccharide: Role of Tumor Necrosis Factor-α

Aggravation of Anti-Myeloperoxidase Antibody-Induced Glomerulonephritis by Bacterial Lipopolysaccharide: Role of Tumor Necrosis Factor-α

Significant IgG-immunoreactivity of the spermatogonia of the germ cell-depleted testis after busulfan treatment

Busulfan kills spermatogonia with the exception of a few that are attached to the basal membrane of the seminiferous epithelium. In mice, these remaining spermatogonia reacted strongly to a goat anti-mouse IgG antibody. Spermatogonia in untreated testes rarely showed the same reactivity. Testicular IgG levels are normally minimal but increase markedly, 4 weeks after busulfan treatment before peaking at week 6. Laser scanning cytometry analysis of control and busulfan-treated testicular cells showed busulfan treatment increased the frequency of cells that were positive for not only IgG (from 0.67 ± 0.29 to 16.5 ± 3.8%) but also for α6-integrin, β1-integrin, GFR(-1 and/or Ret. Thus, an enrichment in putative male stem cells correlates with appearance of IgG expression. Confocal microscopy revealed busulfan-treated cells contained both IgG and GFRα-1, and that the initial surface IgG became intracellular in the weeks following busulfan treatment. The basement membranes of the seminiferous tubules were compromised by busulfan treatment as the mRNA expression profiles of various adhesion molecules in the basement membranes were altered and electron microscopy revealed severe damage. Serum IgG levels increased in a manner corresponding with the increase in testicular IgG levels. Thus, it appears that in the busulfan-treated testis, small breaches of the blood-testis barrier leak IgG that is then taken up by a significant number of spermatogonia. When the busulfan-resistant germ cells were transferred into recipient germ cell-depleted testes, they settled and repopulated the recipient testes. Thus, the IgG-bearing cells observed after busulfan treatment may be putative spermatogonial stem cells.

Functional Role Played by the Glycosylphosphatidylinositol Anchor Glycan of CD48 in Interleukin-18-induced Interferon-γ Production

Interleukin (IL)-18 induces T cells and natural killer cells to produce not only interferon-gamma but also other cytokines by binding to the IL-18 receptor (IL-18R) alpha and beta subunits. However, little is known about how IL-18, IL-18Ralpha, and IL-18Rbeta form a high-affinity complex on the cell surface and transduce the signal. We found that IL-18 and IL-18Ralpha bind to glycosylphosphatidylinositol (GPI) glycan via the third mannose 6-phosphate diester and the second beta-GlcNAc-deleted mannose 6-phosphate of GPI glycan, respectively. To determine which GPI-anchored glycoprotein is involved in the complex of IL-18 and IL-18Ralpha, IL-18Ralpha of IL-18-stimulated KG-1 cells was immunoprecipitated together with CD48 by anti-IL-18Ralpha antibody. More than 90% of CD48 was detected as beta-GlcNAc-deleted GPI-anchored glycoprotein, and soluble recombinant human CD48 without GPI glycan bound to IL-18Ralpha, indicating that CD48 is associated with IL-18Ralpha via both the peptide portion and the GPI glycan. To investigate whether the carbohydrate recognition of IL-18 is involved in physiological activities, KG-1 cells were digested with phosphatidylinositol-specific phospholipase C before IL-18 stimulation. Phosphatidylinositol-specific phospholipase C treatment inhibited the phosphorylation of tyrosine kinases and the following IL-18-dependent interferon-gamma production. These observations suggest that the complex formation of IL-18.IL-18Ralpha. CD48 via both the peptide portion and GPI glycan triggers the binding to IL-18Rbeta, and the IL-18.IL-18Ralpha.CD48.IL-18Rbeta complex induces cellular signaling.

SIRT3, a Mitochondrial Sirtuin Deacetylase, Regulates Mitochondrial Function and Thermogenesis in Brown Adipocytes

SIRT3 is one of the seven mammalian sirtuin homologs of the yeast Sir2 gene, which mediates the effect of caloric restriction on life span extension in yeast and Caenorhabditis elegans. Because adipose tissue is essential in energy homeostasis and also plays a role in life span determination, we decided to investigate the function of sirtuin members in fat. We report here that murine SIRT3 is expressed in brown adipose tissue and is localized on the mitochondria inner membrane. Caloric restriction activates SIRT3 expression in both white and brown adipose. Additionally, cold exposure up-regulates SIRT3 expression in brown fat, whereas elevated climate temperature reduces the expression. Enforced expression of SIRT3 in the HIB1B brown adipocytes enhances the expression of the uncoupling protein PGC-1alpha, UCP1, and a series of mitochondria-related genes. Both ADP-ribosyltransferase and deacetylase activities of SIRT3 are required for this action. Furthermore, the SIRT3 deacetylase mutant exhibits a dominant negative effect by inhibiting UCP1 expression. This inhibitive effect can be abolished by the coexpression of PGC-1alpha, indicating a major role of PGC-1alpha in the SIRT3 action. In addition, SIRT3 stimulates CREB phosphorylation, which reportedly activates PGC-1alpha promoter directly. Functionally, sustained expression of SIRT3 decreases membrane potential and reactive oxygen species production while increasing cellular respiration. Finally, SIRT3, along with genes related to mitochondrial function, is down-regulated in the brown adipose tissue of several genetically obese mice. In summary, our results demonstrate that SIRT3 activates mitochondria functions and plays an important role in adaptive thermogenesis in brown adipose.

Function of Polo-like Kinase 3 in NF-κB-mediated Proapoptotic Response

RelA, the p65 subunit of NF-kappaB transcription factors, plays a key role in regulation of antiapoptotic and proapoptotic responses. However, the downstream target genes regulated by RelA-NF-kappaB in the initiation of proapoptotic signaling were not identified. We previously showed that RelA-NF-kappaB functioned as a proapoptotic factor by activating the p53-signaling pathway in response to doxycycline-induced superoxide. In the present study, we demonstrate that the ability of doxycycline/superoxide to induce expression of polo-like kinase 3 (Plk3) depends on NF-kappaB activity. We identified a kappaB binding site in the promoter of Plk3, and this kappaB site is directly involved in its induction by the RelA-NF-kappaB complex. Plk3 formed a complex with p53 and was involved in the phosphorylation of p53 on Ser-20 in response to superoxide. Inhibition of Plk3 expression by Plk3 small interfering RNA suppressed the doxycycline/superoxide-mediated apoptosis. Overexpression of wild-type Plk3 in HCT116 p53+/+ cells induced rapid apoptosis, whereas overexpression of wild-type Plk3 in HCT116 p53-/- cells and the kinase-defective mutant Plk3(K91R) in p53+/+ cells induced delayed onset of apoptosis. Furthermore, mutagenesis of Plk3 showed that the N-terminal domain (amino acids 1-26) is essential for the induction of delay onset of apoptosis. These data show that Plk3 is a RelA-NF-kappaB-regulated gene that induces apoptosis in both p53-dependent and -independent signaling pathways, suggesting a possible mechanism for RelA-NF-kappaB-regulated proapoptotic responses.

Excretion of Putrescine and Spermidine by the Protein Encoded by YKL174c (TPO5) in Saccharomyces cerevisiae

The properties of the protein encoded by YKL174c (TPO5) were studied. It was found that TPO5 excretes putrescine effectively and spermidine less effectively. Gamma-aminobutyric acid slightly inhibited the excretion of putrescine, but basic amino acids did not affect excretion, suggesting that TPO5 preferentially recognizes polyamines. Accordingly, yeast cells transformed with the plasmid encoding YKL174c (TPO5) were resistant to toxicity caused by 120 mm putrescine or by 3 mm spermidine, and a mutant with a disrupted YKL174c (TPO5) gene was sensitive to toxicity by 90 mm putrescine. The growth of this mutant was faster than that of the wild-type strain. In parallel, there was an increase in putrescine and spermidine content of the YKL174c (TPO5) mutant compared with wild-type. It is noted that TPO5 functions as a suppressor of cell growth by excreting polyamines. The level of YKL174c (TPO5) mRNA was increased by the addition of polyamines to the medium. The degree of induction of the mRNA was spermine > spermidine > putrescine. The subcellular localization of TPO5 was determined by immunostaining of hemagglutinin-tagged TPO5, and it was found on Golgi or post-Golgi secretory vesicles. Excretion of putrescine and spermidine by TPO5 was reduced in cells that have mutations in the secretory or endocytic pathways, indicating that both processes are involved in the excretion of polyamines.

Distinct Conformations of the Corticotropin Releasing Factor Type 1 Receptor Adopted following Agonist and Antagonist Binding Are Differentially Regulated

The corticotropin releasing factor (CRF) type 1 receptor (CRF1) is a class B family G protein-coupled receptor that regulates the hypothalamic-pituitary-adrenal stress axis. Astressin is an amino-terminal truncated analog of CRF that retains high affinity binding to the extracellular domain of the receptor and is believed to act as a neutral competitive antagonist of receptor activation. Here we show that despite being unable to activate the CRF1 receptor, astressin binding results in the internalization of the receptor. Furthermore, entirely different pathways of internalization of CRF1 receptors are utilized following CRF and astressin binding. CRF causes the receptor to be phosphorylated, recruit beta-arrestin2, and to be internalized rapidly, likely through clathrin-coated pits. Astressin, however, fails to induce receptor phosphorylation or beta-arrestin2 recruitment, and internalization is slow and occurs through a pathway that is insensitive to inhibitors of clathrin-coated pits and caveolae. The fate of the internalized receptors also differs because only CRF-induced internalization results in receptor down-regulation. Furthermore, we present evidence that for astressin to induce internalization it must interact with both the extracellular amino terminus and the juxtamembrane domain of the receptor. Astressin binds with 6-fold higher affinity to full-length CRF1 receptors than to a chimeric protein containing only the extracellular domain attached to the transmembrane domain of the activin IIB receptor, yet two 12-residue analogs of astressin have similar affinities for both proteins but are unable to induce receptor internalization. These data demonstrate that agonists and antagonists for CRF1 receptors promote distinct conformations, which are then differentially regulated.

306 ANALYSIS OF GLOBAL DNA METHYLATION IN BOVINE SPERMATOZOA

Methylation of cytosine base in the CpG dinucleotide sequence (DNA methylation) is a major epigenetic modification of the genome and plays an important role in gene expression. Recently, global DNA methylation in genome was studied by using a restriction landmark genomic scanning (RLGS) method and/or a representational difference analysis (RDA) method. However, these methods are complicated and need to use restriction enzymes. Therefore, the information derived from those methods is restricted to the region of the DNA sequence which is able to be cleaved by restriction enzymes. In this study, to establish a simple method to estimate global DNA methylation level in bovine spermatozoa, we tried to develop the DNA methylation analyzing method by using immunostaining of 5-methylcytosine. The immunostaining method for 5-methylcytosine in this study was based on the method developed by Benchaib et al. (2003 Fertil. Steril. 80, 947-952) for human spermatozoa. Because of the species difference, we modified some treatments to apply to bovine spermatozoa. Frozen-thawed bovine spermatozoa were washed by using 30 and 45% Percoll gradient solutions. After washing, spermatozoa were treated with 0.25 dithicthreitol M (DTT) and 1% sodium dodecyl sulfate (SDS) at room temperature (RT). Then, treated spermatozoa were spread on a slide glass with Cytospin4 (30g, 5 � 104 cells/mL) and air-dried at RT. Air-dried bovine spermatozoa specimens were fixed in methanol: glacial acetic acid (3:1) solution at RT and treated with 1% Triton X and 1% SDS at RT; DNA was denatured with 6 N HCl at RT. After the denaturation, 5-methylcytosine in sperm DNA was analyzed by immunofluorescence technique with mouse anti 5-methylcytosine antibody and fluorescein isothiocyanate (FITC)-conjugated goat anti mouse IgG antibody. The total sperm DNA was counterstained with propidium iodide (PI). Stained samples were observed with a confocal laser scanning microscope and obtained images were analyzed with fluorescence image analysis software. The area that was clearly stained with PI in each sperm head was designated and measured as the area of total sperm DNA, and the number of the dots that showed FITC fluorescence within the total sperm DNA area was designated and measured as the area of 5-methylcytosine in total sperm DNA. The area measurement was performed with fixed light strength. Three bovine spermatozoa samples derived from different bulls, used daily for calf production by AI, were examined. The ratio of the mean total area of the 5-methylcytosine in sperm DNA to the mean total area of the sperm DNA was 34.1% in bull A (9.13 � 5.66 �m2, 26.75 � 5.29 �m2, n = 57), 45.2% in bull B (16.60 � 3.79 �m2, 36.74 � 5.95 �m2, n = 41) and 43.9% in bull C (14.66 � 4.27 �m2, 33.45 � 7.13 �m2, n = 22). There was significant difference in the ratio between bull A and bulls B and C (P < 0.01). More research is required to evaluate the meaning of this individual difference of DNA methylation between bulls. This work was supported by Wakayama Prefecture Collaboration of Regional Entities for the Advanced of Technological Excellence, Japan, and by a Grant-in-Aid for the 21st Century Center of Excellence Program of the MEXT, Japan.

Biochemical Differences in the αβ T Cell Receptor·CD3 Surface Complex between CD8+ and CD4+ Human Mature T Lymphocytes

We have reported the existence of biochemical and conformational differences in the alphabeta T cell receptor (TCR) complex between CD4(+) and CD8(+) CD3gamma-deficient (gamma(-)) mature T cells. In the present study, we have furthered our understanding and extended the observations to primary T lymphocytes from normal (gamma(+)) individuals. Surface TCR.CD3 components from CD4(+) gamma(-) T cells, other than CD3gamma, were detectable and similar in size to CD4(+) gamma(+) controls. Their native TCR.CD3 complex was also similar to CD4(+) gamma(+) controls, except for an alphabeta(deltaepsilon)(2)zeta(2) instead of an alphabetagammaepsilondeltaepsilonzeta(2) stoichiometry. In contrast, the surface TCRalpha, TCRbeta, and CD3delta chains of CD8(+) gamma(-) T cells did not possess their usual sizes. Using confocal immunofluorescence, TCRalpha was hardly detectable in CD8(+) gamma(-) T cells. Blue native gels (BN-PAGE) demonstrated the existence of a heterogeneous population of TCR.CD3 in these cells. Using primary peripheral blood T lymphocytes from normal (gamma(+)) donors, we performed a broad epitopic scan. In contrast to all other TCR.CD3-specific monoclonal antibodies, RW2-8C8 stained CD8(+) better than it did CD4(+) T cells, and the difference was dependent on glycosylation of the TCR.CD3 complex but independent of T cell activation or differentiation. RW2-8C8 staining of CD8(+) T cells was shown to be more dependent on lipid raft integrity than that of CD4(+) T cells. Finally, immunoprecipitation studies on purified primary CD4(+) and CD8(+) T cells revealed the existence of TCR glycosylation differences between the two. Collectively, these results are consistent with the existence of conformational or topological lineage-specific differences in the TCR.CD3 from CD4(+) and CD8(+) wild type T cells. The differences may be relevant for cis interactions during antigen recognition and signal transduction.

CK2 Phosphorylation of the Armadillo Repeat Region of β-Catenin Potentiates Wnt Signaling

Protein kinase CK2 is a ubiquitous serine/threonine kinase involved in many biological processes. It is overexpressed in many malignancies including rodent and human breast cancer, and is up-regulated in Wnt-transfected mammary epithelial cells, where it can be found in a complex with dishevelled and beta-catenin. beta-Catenin is a substrate for CK2 and inhibition of CK2 reduces levels of beta-catenin and dishevelled. Here we report that inhibition of CK2 using pharmacologic agents or expression of kinase inactive subunits reduces beta-catenin-dependent transcription and protein levels in a proteasome-dependent fashion. The major region of phosphorylation of beta-catenin by CK2 is the central armadillo repeat domain, where carrier proteins like axin and the adenomatous polyposis coli gene product APC interact with beta-catenin. The major CK2 phosphorylation site in this domain is Thr393, a solvent-accessible residue in a key hinge region of the molecule. Mutation of this single amino acid reduces beta-catenin phosphorylation, cotranscriptional activity, and stability. Thus, CK2 is a positive regulator of Wnt signaling through phosphorylation of beta-catenin at Thr393, leading to proteasome resistance and increased protein and co-transcriptional activity.

P63 Coordinates Anogenital Modeling and Epithelial Cell Differentiation in the Developing Female Urogenital Tract

p63 is a p53 homologue required for cutaneous development that is expressed in immature squamous epithelium and reserve cells of the cervix. Humans with p63 mutations exhibit defects in limb, accessory organ (skin appendage, breast, prostate), and genitourinary development. Because p63 expression patterns imply a strong role of the gene in the female genital tract development, newborn female p63-/-, +/-, and +/+ mice were examined in situ, dissected, and compared. Nuclear p63 protein was localized to the skin, vagina, bladder, urethra, and basal columnar cells of the caudal uterus in p63+/+ and +/- animals. p63-/- mice exhibited abnormal genital morphogenesis with hypoplastic genitalia, a single cloacal opening, and persistence of columnar epithelium at lower genital tract sites that normally undergo squamous and urothelial differentiation. The defects observed support p63-dependent pathways of genital tract development that permit externally, ectodermal basal cell replenishment integral to reciprocal epithelial stromal signaling, urorectal septation, and modeling of the external genitalia; and internally, the emergence of basal epithelial cell populations capable of divergent epithelial cell differentiation in the vagina, cervix, and urinary tract. Defects in the first pathway explain imperforate anus, vaginal septum, genital hypoplasia, and micropenis reported in humans with p63 mutations. The second is necessary for the generation of multipotential reserve cells in the cervix and may be operative in other epithelial stromal interactions integral to the emergence of uterine basal cells later in life.