Modeling breast cancer in vivo and ex vivo reveals an essential role of Pin1 in tumorigenesis

Phosphorylation on certain Ser/Thr-Pro motifs is a major oncogenic mechanism. The conformation and function of phosphorylated Ser/Thr-Pro motifs are further regulated by the prolyl isomerase Pin1. Pin1 is prevalently overexpressed in human cancers and implicated in oncogenesis. However, the role of Pin1 in oncogenesis in vivo is not known. We have shown that Pin1 ablation is highly effective in preventing oncogenic Neu or Ras from inducing cyclin D1 and breast cancer in mice, although it neither affects transgene expression nor mammary gland development. Moreover, we have developed an ex vivo assay to uncover that a significant fraction of primary mammary epithelial cells from Neu or Ras mice display various malignant properties long before they develop tumors in vivo. Importantly, these early transformed properties are effectively suppressed by Pin1 deletion, which can be fully rescued by overexpression of cyclin D1. Thus, Pin1 is essential for tumorigenesis and is an attractive anticancer target. Our ex vivo assay can be used to study early events of breast cancer development in genetically predisposed mice.     

Binding potency of peptide fragments of type 1 collagen cross-linked N-telopeptide measured by an enzyme-linked immunosorbant assay

Osteoporosis represents a major healthcare problem affecting elderly person. Urinary level of the crosslinked N-telopeptide of type I collagen is a sensitive marker of bone resorption. Ten overlapping peptides covering the N-telopeptide of alpha-2 type I collagen were synthesized, purified, and assayed for their relative binding response to anti-type I collagen cross-linked N-telopeptide (NTX) antibody by using a competitive-inhibition enzyme-linked immunosorbent assay (ELISA). Peptides 1, 2, and 3, containing the N-terminal sequence of N-telopeptide, showed higher binding potency than peptides 4-10, suggesting that these peptides may contain binding sites for anti-NTX antibodies, and can serve as the lead for further preparation of their antibodies in order to develop novel bioassays for monitoring the bone loss in humans.     

Skin Autofluorescence Is Associated with Endothelial Dysfunction in Uremic Subjects on Hemodialysis

Background

Elevated levels of advanced glycation end products (AGEs) within tissues may contribute to endothelial dysfunction, an early indicator of atherosclerosis. We aimed to investigate whether levels of skin AGEs could be a useful marker to predict endothelial dysfunction in uremic subjects on hemodialysis.

Methods and Results

One hundred and nineteen uremic patients on hemodialysis and 57 control subjects with moderate-to-high cardiovascular risk factors and without chronic kidney disease (CKD) were enrolled. We used ultrasound to measure flow-mediated vasodilation (FMD). An AGE reader measured skin autoflurorescence (AF). We then compared differences in FMD and skin AF values between the two groups. The uremic subjects had significantly higher levels of skin AF (3.47±0.76 AU vs. 2.21±0.45 arbitrary units; P<0.01) and significantly lower levels of FMD (4.79%±1.88% vs. 7.19%±2.17%; P<0.01) than the non-CKD subjects. After adjusting for all potential covariates, we found that skin AF level independently predicted FMD in both the hemodialysis and the non-CKD groups. In the hemodialysis group, skin AF ≥ 3.05 arbitrary units predicted abnormal FMD at a sensitivity of 87.9% and a specificity of 78.6% (P<0.01).

Conclusions

Skin AF could be a useful marker to predict endothelial dysfunction in uremic subjects on hemodialysis.     

Genetically engineered biosynthesis of macrolide derivatives including 4-amino-4,6-dideoxy-L-glucose from Streptomyces venezuelae YJ003-OTBP3

Two sugar biosynthetic cassette plasmids were used to direct the biosynthesis of a deoxyaminosugar. The pOTBP1 plasmid containing TDP-glucose synthase (desIII), TDP-glucose-4,6-dehydratase (desIV), and glycosyltransferase (desVII/desVIII) was constructed and transformed into S. venezuelae YJ003, a strain in which the entire gene cluster of desosamine biosynthesis is deleted. The expression plasmid pOTBP3 containing 4-aminotransferase (gerB) and 3,5-epimerase (orf9) was transformed again into S. venezuelae YJ003- OTBP1 to obtain S. venezuelae YJ003-OTBP3 for the production of 4-amino-4,6-dideoxy-L-glucose derivatives. The crude extracts obtained from S. venezuelae ATCC 15439, S. venezuelae YJ003, and S. venezuelae YJ003-OTBP3 were further analyzed by TLC, bioassay, HPLC, ESI/MS, LC/MS, and MS/MS. The results of our study clearly shows that S. venezuelae YJ003-OTBP3 constructs other new hybrid macrolide derivatives including 4-amino-4,6-dideoxy-L-glycosylated YC-17 (3, [M+ Na+] m/z=464.5), methymycin (4, m/z=480.5), novamethymycin (6, m/z=496.5), and pikromycin (5, m/z=536.5) from a 12- membered ring aglycon (10-deoxymethynolide, 1) and 14-membered ring aglycon (narbonolide, 2). These results suggest a successful engineering of a deoxysugar pathway to generate novel hybrid macrolide derivatives, including deoxyaminosugar.     

Assembly of the SIR complex and its regulation by O-acetyl-ADP-ribose, a product of NAD-dependent histone deacetylation

Assembly of silent chromatin domains in budding yeast involves the deacetylation of histone tails by Sir2 and the association of the Sir3 and Sir4 proteins with hypoacetylated histone tails. Sir2 couples deacetylation to NAD hydrolysis and the synthesis of a metabolite, O-acetyl-ADP-ribose (AAR), but the functional significance of NAD hydrolysis or AAR, if any, is unknown. Here we examine the association of the Sir2, Sir3, and Sir4 proteins with each other and histone tails. Our analysis reveals that deacetylation of histone H4-lysine 16 (K16), which is critical for silencing in vivo, is also critical for the binding of Sir3 and Sir4 to histone H4 peptides in vitro. Moreover, AAR itself promotes the association of multiple copies of Sir3 with Sir2/Sir4 and induces a dramatic structural rearrangement in the SIR complex. These results suggest that Sir2 activity modulates the assembly of the SIR complex through both histone deacetylation and AAR synthesis.     

Involvement of 15-lipoxygenase in the inflammatory arthritis

15-Lipoxygenase (15-LOX) is involved in many pathological processes. The aim of this study is to examine the role of 15-LOX in the matrix metalloproteinase (MMP) expression and inflammatory arthritis. It was found that treatment of 15-LOX downstream product of 15-(S)-HETE (15-S-hydroxyeicosatetraenoic acid) increased the mRNA and protein levels of MMP-2 in rheumatoid arthritis synovial fibroblast (RASF) derived from rheumatoid arthritis patients. The enhancement effect of 15-(S)-HETE was antagonized by the addition of LY294002 (PI3K inhibitor) and PDTC (NF-κB inhibitor). Treatment of 15-(S)-HETE increased the phosphorylation of AKT, nuclear translocation of p65 and the breakdown of IκBα. TNF-α and IL-1β are the key cytokines involved in arthritis and also increase the activity of MMP-2 in RASF, which was antagonized by pretreatment with 15-LOX inhibitor PD146176 or knockdown of 15-LOX. It was also found that these two cytokines increased the expression of 15-LOX in RASF. Treatment of glucocorticoid but not NSAIDs inhibited 15-(S)-HETE-induced expression of MMP-2. In comparison with wild-type mice, adjuvant-induced arthritis and MMP-2 expression in synovial membrane were markedly inhibited in 15-LOX knockout (KO) mice. These results indicate that 15-LOX plays an important role in the disease progression of arthritis and may be involved in the inflammatory action induced by TNF-α and IL-1β. 15-LOX is thus a good target for developing drugs in the treatment of inflammatory arthritis.     

A chimeric mouse-human antibody that retains specificity for HIV gp120 and mediates the lysis of HIV-infected cells

Murine mAb BAT123, which was made against the envelope glycoprotein gp120 of HTLV-IIIB strain of HIV type 1 (HIV-1), is capable of neutralizing HTLV-IIIB in vitro. It also inhibits the fusion between uninfected CD4+ cells and HIV-1-infected cells to form syncytia. As a step to explore the potential utility of the anti-HIV antibody in vivo, we have constructed a mouse-human chimeric antibody by rDNA techniques. The chimeric antibody, which bears the variable domains of mouse antibody BAT123 and constant domains Cr1 and C kappa of human Ig retains the Ag specificity of BAT123 as determined by its reactivity with HIV-1-infected H9 cells, gp120 in Western blot analysis, and the oligopeptide recognized by BAT123. The antiviral activities of the chimeric antibody in neutralizing HIV-1 infection as well as inhibiting the syncytia formation are also found identical to those of the parent murine antibody. Moreover, in the presence of human blood mononuclear cells, the chimeric antibody but not BAT123 (mouse IgG1) induces antibody-dependent cellular cytotoxicity. The findings point to the potential usefulness of the chimeric antibody in treating patients infected with HIV-1.     

Genetics of divergence in male wing pigmentation and courtship behavior between Drosophila elegans and D. gunungcola

Many sex-specific traits involved in mating consist of functionally coordinated morphologies and behaviors. How the components of these complex traits evolve and become coordinated during evolution is unknown. In order to understand how such trait complexes evolve and diversify, we must decipher the genetic underpinnings of their components. In this study, we begin to elucidate the genetic architecture underlying differences in functionally related male pigmentation and behavior between two Asian Drosophila melanogaster group species, D. elegans and D. gunungcola. D. elegans possesses a male-specific wing melanin spot and a stereotypical wing display element in male courtship, whereas D. gunungcola lacks both of these traits. Using reciprocal F1 male hybrids, we demonstrate that the X-chromosome contains a major locus or loci required for wing spot formation and that autosomal loci largely determine the male courtship display. Using phenotypic and genetic analysis of backcross progeny, we further demonstrate that both the wing spot and courtship differences between the two species are polygenic and both depend at least in small part on genetic factors on both the X and the autosomes. Finally, we find that male wing spot size and courtship wing display are highly correlated in backcross progeny, suggesting that linkage or pleiotropy may have been involved in their coordinated evolution.