We investigated the immunohistochemical localization of androgen receptor (AR) using a polyclonal antibody for 55 KD recombinant human AR in human tissues fixed with 4% paraformaldehyde solution and embedded in paraffin. Immunoreactive AR was restricted to the nuclei of various tissues. Among the well-known androgen target organs, secretory cells and basal cells of the prostate, spermatogonia, spermatocytes, Sertoli cells and Leydig cells of the testis, epithelial cells of the rete testis, fibroblasts in the whole organ, squamous cells, sweat gland and hair follicle cells of the skin, and hepatocytes of the liver were positive for AR. In addition, smooth muscle cells of the prostate, uterus, urinary bladder, gastrointestinal tract, arteries, and arterioles were strongly immunoreactive for AR. Cardiac muscle and striated muscle of psoas were positive for AR. Acinar cells, ductal cells, and myoepithelial cells of the breast, endocervical and endometrial cells of the uterus, cyto- and syncytiotrophoblast of the chorionic villi, and tubules of the kidney were also positive for AR. Most FSH, LH, and some GH endocrine cells in the anterior and posterior lobes of the pituitary gland, follicular cells of the thyroid gland, and adrenocortical cells were positive for AR. Cells immunoreactive for AR were ubiquitously distributed throughout the entire body. The present study demonstrated the diversity of androgen effects on many target tissues.

MAIL (molecule-possessing ankyrin repeats induced by lipopolysaccharide) is a nuclear IB protein that is also termed interleukin-1-inducible nuclear ankyrin repeat protein or inhibitor of nuclear factor B (IB) . In this study, we generated Mail ؊/؊ mice to investigate the roles of MAIL in whole organisms. Mail ؊/؊ mice grew normally until 4 -8 weeks after birth, when they began to develop lesions in the skin of the periocular region, face, and neck. MAIL mRNA and protein were constitutively expressed in the skin of wild type controls, especially in the keratinocytes. Serum IgE was higher in Mail ؊/؊ mice than in normal. Histopathological analysis indicated that the Mail ؊/؊ skin lesions appeared to be atopic dermatitis (AD) eczema with inflammatory cell infiltration. In addition, markedly elevated expression of some chemokines such as thymus and activation-regulated chemokine was detected in the Mail ؊/؊ skin lesions, similar to that observed in the skin of patients with AD. In Mail ؊/؊ mice, MAIL-deficient keratinocytes might be activated to produce chemokines and induce intraepidermal filtration of inflammatory cells, resulting in the onset of the AD-like disease. These findings suggest that MAIL is an essential molecule for homeostatic regulation of skin immunity. The Mail ؊/؊ mouse is a valuable new animal model for research on AD.

No abstract

a b s t r a c tThe breast cancer susceptibility protein BRCA2 is essential for recombinational DNA repair. BRCA2 specifically binds to RAD51 via eight BRC repeat motifs and delivers RAD51 to double-stranded DNA breaks. In this study, a mammalian two-hybrid assay and competitive ELISA showed that the interaction between BRC repeat 4 (BRC4) and RAD51 was strengthened by the substitution of a single BRC4 amino acid from valine to isoleucine (V1532I). However, the cancer-associated V1532F mutant exhibited very weak interaction with RAD51. This study used a comparative analysis of BRC4 between animal species to identify V1532 as an important residue that interacts with RAD51. Structured summary of protein interactions:cRAD51 physically interacts with cRAD51 by two hybrid (View interaction) fBRC4 physically interacts with cRAD51 by two hybrid (View interaction) cBRC4 physically interacts with cRAD51 by two hybrid (View interaction) hBRC4 physically interacts with hBRC4 and hRAD51 by competition binding (View Interaction 1, 2) hBRC4 physically interacts with cRAD51 by two hybrid (View interaction) hBRC4 binds to hRAD51 by enzyme linked immunosorbent assay (View interaction)

In humans, mutations of the breast cancer susceptibility protein BRCA2 interact with recombinase RAD51 and increase the risk of cancer. This interaction occurs via a series of eight BRC repeat sequences in BRCA2. A mammalian two-hybrid assay using individual BRC repeats demonstrated that all the repeats except BRC6 bind RAD51 strongly (BRC1, 2 and 4), with intermediate strength (BRC8), or weakly (BRC3, 5 and 7). In serial deletion mutation experiments, the binding strengths were increased when the C-terminal BRC repeat was removed from BRC1-8, BRC1-5 and BRC1-3. These results provide an understanding of the basic function of canine BRCA2 and may be helpful to estimate the effect of missense or truncation mutations in canine mammary tumours

In vitro cell studies might be a useful tool for studying tendon pathology, but no suitable in vitro models exist for tendon disorders. The purpose of this study was to confirm whether cell scratch culture using tendon-derived fibroblasts can provide a suitable in vitro tendon disorder model. Extracellular matrix components were examined immunohistochemically in tendon tissue, and then their related gene expression levels were analyzed by conventional reverse transcription polymerase chain reaction (RT-PCR) and/or quantitative real-time RT-PCR in tissues and cells. Collagen type I (Col I), collagen type III (Col III), tenascin-C (TN-C) and cartilage oligomeric matrix protein (COMP) were detected in tendon tissue sections, and RT-PCR confirmed their expression in tendon tissue and cells. Cells that had been cultured from explanted tendon tissue maintained the characteristics of in vivo tendon cells. The combination of TN-C and COMP might be a useful marker of tendon cells because they display more tendon-specific expression than Col I and III. In particular, the significant increase of TN-C mRNA expression in the scratch wound assay, at 12 hr after scratching, concomitant with the regeneration of the cell sheet, indicates its crucial role in tendon cell proliferation and migration. Thus, TN-C appears to be a key factor in tendon wound healing. In vitro cell scratch assays using tendon cells appear to mimic the repair of tendon tissue after injury.

ABSTRACT. It has recently been suggested that the chemokine receptor CXCR4 and its ligand SDF-1 (CXCL12) promote metastasis of various cancers in humans. Since feline mammary tumors also metastasize to distant organs frequently, we used real-time quantitative PCR to examine the expression of feline CXCR4 (fCXCR4) in ten feline mammary tumor cell lines and seven feline mammary tumor tissues, and also the expression of feline SDF-1 (fSDF-1) in various organs. Cell lines derived from metastatic regions expressed more fCXCR4 than those derived from primary tumors. Mammary tumor tissues overexpressed more fCXCR4 than normal mammary tissues. Organs with high levels of fSDF-1 expression represent common sites of metastasis. Migration assays using the feline mammary tu mor cell line NAC were also performed to test the activity of TN14003 and TC14012, antagonists of human CXCR4, to antagonize fCXCR4 expressed on NAC cells. TN14003 and TC14012 inhibited migration of NAC cells. We conclude that fCXCR4 may be a therapeutic target for feline mammary tumors. KEY WORDS: CXCR4, feline, mammary tumor, metastasis, SDF-1.

ABSTRACT. Molecule possessing ankyrin-repeats induced by lipopolysaccharide (MAIL) is a nuclear IκB protein that is also known as interleukin-1-inducible nuclear ankyrin repeat protein and inhibitor of nuclear factor κBζ (IκBζ). We previously observed that MAILdeficient mice were affected by atopic dermatitis-like skin lesions and demonstrated the importance of MAIL in the skin. In this study, we investigated MAIL expression in mouse keratinocytes. MAIL mRNA was constitutively expressed in the skin epidermis. MAIL expression was also confirmed in primary keratinocytes and the PAM212 keratinocyte cell line. The inhibitors of nuclear factor κB (NF-κB)-Bay11-7082 and the IκBαM supersuppressor-considerably downregulated MAIL expression in the keratinocytes. Immunoreactivity for NF-κB components was localized in the cytoplasm and nucleus of normal unstimulated keratinocytes. The expression level of MAIL in the skin did not change following lipopolysaccharide (LPS) administration to mice. Interestingly, in accordance with the in vivo findings, the MAIL expression level did not change following LPS stimulation even in primary keratinocytes; however, MAIL expression was strongly increased by interleukin-1 stimulation. These results collectively suggest that the constitutive expression of MAIL in keratinocytes is controlled, at least in part, by NF-κB and that there may be LPS-specific repressive mechanisms that inhibit MAIL induction.