Sulfated glycosaminoglycans mediate prion-like behavior of p53 aggregates

Sulfated glycosaminoglycans (GAGs) such as heparan sulfate (HS) are heteropolysaccharides implicated in the pathology of protein aggregation diseases including localized and systemic forms of amyloidosis. Among subdomains of sulfated GAGs, highly sulfated domains of HS, called HS S-domains, have been highlighted as being critical for HS function in amyloidoses. Recent studies suggest that the tumor suppressor p53 aggregates to form amyloid fibrils and propagates in a prion-like manner; however, molecules and mechanisms that are involved in the prion-like behavior of p53 aggregates have not been addressed. Here, we identified sulfated GAGs as molecules that mediate prion-like behavior of p53 aggregates. Sulfated GAGs at the cell surface were required for cellular uptake of recombinant and cancer cell-derived p53 aggregates and extracellular release of p53 from cancer cells. We further showed that HS S-domains accumulated within p53 deposits in human ovarian cancer tissues, and enzymatic remodeling of HS S-domains by Sulf-2 extracellular sulfatase down-regulated cellular uptake of p53 aggregates. Finally, sulfated GAG-dependent cellular uptake of p53 aggregates was critical for subsequent extracellular release of the aggregates and gain of oncogenic function in recipient cells. Our work provides a mechanism of prion-like behavior of p53 aggregates and will shed light on sulfated GAGs as a common mediator of prions.

Glycosaminoglycans (GAGs) are linear, unbranched polysaccharides that are attached to a core protein to form proteoglycans. The GAG chain structure is heterogeneous and can vary in net charges. Sulfated GAGs, such as heparan sulfate (HS) and chondroitin sulfate (CS), consist of repeating disaccharide units of a uronic acid and either N-acetylglucosamine or N-acetylgalactosamine. The structural diversity of GAGs confers binding selectivity of GAGs to their ligands and, thereby, plays an essential part in the functions of proteoglycans including organogenesis, signal transduction, and inflammation. Sulfated GAGs have also been implicated in the pathology of several diseases such as cancer and amyloidosis, which is the most common protein aggregation disease (1–3). Since the identification of sulfated GAGs as a common constituent of amyloid deposits in systemic and localized amyloidoses (4), sulfated GAGs have been established to affect the pathogenesis and progression of amyloidoses by promoting the formation and cellular interaction of protein aggregates (1).The human TP53 gene encodes a nuclear tumor suppressor—p53—that can also respond to several stress conditions to induce cell cycle arrest and apoptosis (5). TP53 gene mutations occur in more than 50% of human cancers, which makes it the most common mutant gene in cancers. The DNA-binding domain of p53 is conformationally unstable (6), and several hotspot mutants are reportedly unfolded (7). p53 was recently reported to form protein aggregates in human cancer tissues and in vitro (8–11), which suggests that p53-mutant cancers may be a new class of protein aggregation diseases. p53 aggregates were proposed to demonstrate prion-like behavior like that of many other protein aggregates (12). We and others reported that the patterns and degrees of GAG’s sulfation modification (13–16), especially the highly sulfated domains of HS (HS S-domains), are essential for the pathology and progression of systemic and localized amyloidoses. However, the involvement of sulfated GAGs in transcellular propagation of p53 aggregates must still be elucidated.Ovarian cancer is often diagnosed at an advanced stage, and little progress has been achieved in chemotherapy treatment (17). Alterations in the TP53 gene are quite prevalent in ovarian cancer, especially in the most common histological subtype—high-grade serous ovarian carcinoma (96%) (18)—which supports the pathological significance of p53 mutations. In our study here, we investigated the accumulation of HS S-domains in ovarian cancer tissues and the involvement of sulfated GAGs in extracellular release and cellular interaction of p53 aggregates. HS S-domains can be postsynthetically remodeled by Sulf-2 extracellular sulfatase (19). In addition, we studied whether enzymatic remodeling of HS S-domains would affect cellular uptake of extracellularly released p53. Our study supports the common roles of GAGs and their sulfation modifications in transcellular propagation of various protein aggregates.     

Topical ER36009, a RARgamma-selective retinoid, decreases abdominal white adipose tissue and elicits changes in expression of genes related to adiposity and thermogenesis

Chronic topical treatment of rats with a new RARγ-selective retinoid, ER36009, resulted in a significant reduction of epididymal white adipose tissue and a significant increase of interscapular brown adipose tissue without affecting food intake. ER36009 markedly decreased PPARγ, 11β-HSD1, and Bcl-2 mRNA levels, and increased Bax mRNA in white adipose tissue, while it upregulated UCP1 and UCP3 mRNAs in brown adipose tissue and UCP3 mRNA in gastrocnemial muscle. These results suggest that ER36009 has multiple effects on adipose tissue biology and the energy balance. Topically applied ER36009 may have potential for the treatment of obesity.     

TLR7-dependent and FcγR-independent production of type I interferon in experimental mouse lupus

Increased type I interferon (IFN-I) production and IFN-stimulated gene (ISG) expression are linked to the pathogenesis of systemic lupus erythematosus (SLE). Although the mechanisms responsible for dysregulated IFN-I production in SLE remain unclear, autoantibody-mediated uptake of endogenous nucleic acids is thought to play a role. 2,6,10,14-tetramethylpentadecane (TMPD; also known as pristane) induces a lupus-like disease in mice characterized by immune complex nephritis with autoantibodies to DNA and ribonucleoproteins. We recently reported that TMPD also causes increased ISG expression and that the development of the lupus is completely dependent on IFN-I signaling (Nacionales, D.C., K.M. Kelly-Scumpia, P.Y. Lee, J.S. Weinstein, R. Lyons, E. Sobel, M. Satoh, and W.H. Reeves. 2007. Arthritis Rheum. 56:3770–3783). We show that TMPD elicits IFN-I production, monocyte recruitment, and autoantibody production exclusively through a Toll-like receptor (TLR) 7– and myeloid differentiation factor 88 (MyD88)–dependent pathway. In vitro studies revealed that TMPD augments the effect of TLR7 ligands but does not directly activate TLR7 itself. The effects of TMPD were amplified by the Y-linked autoimmune acceleration cluster, which carries a duplication of the TLR7 gene. In contrast, deficiency of Fcγ receptors (FcγRs) did not affect the production of IFN-I. Collectively, the data demonstrate that TMPD-stimulated IFN-I production requires TLR7/MyD88 signaling and is independent of autoantibody-mediated uptake of ribonucleoproteins by FcγRs.     

Cross-linking of B cell antigen receptor-related structure of pre-B cell lines induces tyrosine phosphorylation of p85 and p110 subunits and activation of phosphatidylinositol 3-kinase

To understand the function of B cell antigen receptor (BCR)-relatedcomplex on pre-B cells (pre-BCR, V_preB5/µ heavy chain/lg-/lg-ß),we examined pre-BCR- and BCR-mediated signaling events in humanand mouse pre-B (Nalm-6, 697, NFS-5), Immature B (lgM⁺ Daudi,WEH1–231) and mature B (lgM⁺;lgD⁺ BALL1) cell lines. Antl-µcross-linking induced tyrosine phosphorylation of the cytoplasmicproteins In each cell type, but did not induce a detectableCa²⁺ mobilization response in pre-B cells. While the pre-B cellsexpressed Syk protein at levels similar to those found In Bcell lines, pre-BCR cross-linkage did not Induce phosphorylationof Syk tyrosine residues. Different protein kinase C Isozymeswere expressed by pre-B (PKC-), Immature B (PKC- and -ß)and mature B (PKC-ß) cell lines. Anti-µ cross-linkingInduced PKC translocatlon from the cytosolic to the membranecompartment In Immature and mature B cells, but did not havethis effect In a pre-B cell line. Antl-µ cross-linkinginduced tyrosine phosphorytation of the p85 and p110 subunitsof phophatldylinositol 3-kinase (PI3-kinase) In both pre-B andB cell lines, but the pre-BCR induced PI3-kinase activationwas Syk independent. Ligation of the pre-BCR complex thus triggersa characteristic signaling pattern In pre-B cells.     

The serum leptin level and body mass index in Melanesian and Micronesian Solomon Islanders: Focus on genetic factors and urbanization

Objectives: This study examined the association between the serum leptin level and body mass index (BMI) and the effects of urbanization and polymorphisms of leptin (LEP) or leptin receptor (LEPR) genes on the leptin level in three Solomon Islands populations. Methods: A Melanesian population living in a remote area (participants: 106 males and 106 females, ages: 18–74 years), a Melanesian population in an urban area (89 and 94, 18–79 years), and a Micronesian population who migrated to a peri‐urban area in the 1960s (84 and 69, 18–71 years) were studied. Anthropometric and serum leptin measurements and genotyping for LEP G‐2548A and LEPR K109R and Q223R were performed. Results: The prevalence of obesity (BMI ≥ 30 kg/m²) was the highest in the Micronesian population (30.1%), followed by the urban (18.6%) and the rural (2.4%) Melanesian population. The serum leptin concentration was the highest in the urban Melanesian, followed by the Micronesian and the rural Melanesian populations (P < 0.05). Interestingly, the parameter coefficients of the leptin concentrations on the BMIs were nearly identical in the urban and rural Melanesians after adjusting for age and gender. The LEPR 223Q/Q genotype was associated with an increased leptin level only in the Micronesian population after adjusting for BMI (P = 0.0008 and 0.0016 referenced to the Q/R and the R/R types, respectively). Conclusions: These observations suggest that the increase in obesity in the Micronesians had a genetic component while that in Melanesians might have been related with the urbanization. Am. J. Hum. Biol., 2011. © 2010 Wiley‐Liss, Inc.     

Non-mass-like enhancement on contrast-enhanced breast MR imaging: Lesion characterization using combination of dynamic contrast-enhanced and diffusion-weighted MR images

Purpose

To evaluate the diagnostic accuracy of a combination of dynamic contrast-enhanced MR imaging (DCE-MRI) and diffusion-weighted MR imaging (DWI) in characterization of lesions showing non-mass-like enhancement on breast MR imaging and to find the strongest discriminators between carcinoma and benignancy.

Materials and methods

We analyzed consecutive MR images in 45 lesions showing non-mass like enhancement in 41 patients. We analyzed lesion size, distribution, internal enhancement, kinetic curve pattern, and apparent diffusion coefficient (ADC) values. We applied univariate and multivariate analyses to find the strongest indicators for malignancy. In a validation study, 22 non-mass-like enhancement lesions in 21 patients were examined. We calculated diagnostic accuracy when we presume category 4b, 4c, and 5 lesions as malignant or high to moderate suspicion for malignancy, and category 4a and 3 as low suspicion for malignancy or benign.

Results

Segmental distribution (P = 0.018), clumped internal enhancement (P = 0.005), and ADC less than 1.3 × 10⁻³ mm²/s (P = 0.047) were the strongest MR indicators of malignancy. In a validation study, sensitivity, specificity, positive predictive value, negative predictive value and accuracy were 87% (13/15), 86% (6/7), 93% (13/14), 75% (6/8) and 86% (19/22), respectively.

Conclusion

The combination of DCE-MRI and DWI showed high diagnostic accuracy in characterization of non-mass-like enhancement lesions on breast MR images.     

Is parenchyma-preserving hepatectomy a noble option in the surgical treatment for high-risk patients with hilar bile duct cancer?

BACKGROUND: The essential minimum of hepatic segmentectomy combined with caudate lobectomy (parenchyma-preserving hepatectomy) has been recommended particularly for high-risk patients with hilar bile duct cancer to minimize the risk of postoperative liver failure. This quality control study investigated whether parenchyma-preserving hepatectomy is a "noble option" in the surgical treatment of hilar bile duct cancer. PATIENTS AND METHODS: A total of 53 patients with hilar bile duct cancer underwent surgical resection. These patients were retrospectively classified into a major hepatectomy group (major Hx, n=30), a parenchyma-preserving hepatectomy group (preserving Hx, n=11), and a hilar bile duct resection group (HBDR, n=12). A preserving Hx consisted of caudate lobectomy, either alone (n=3), or combined with resection of segment 4 (S4, n=4), or S58 (n=3) or S458 (n=1). The preserving Hx was used for high-risk patients in whom tumor tissue was diagnosed to be Bismuth type I and II by preoperative selective percutaneous transhepatic cholangiography. RESULTS: The mean numbers of hepatico-jejunostomies were 2.8, 4.8, and 4.6 in the respective groups. Mortality rates including hospital death were 13.3%, 0%, and 0% respectively. Morbidity rates were 46.7%, 54.5%, and 33.3%. The preserving Hx group encountered no liver failure (T.Bil>10 mg/dl, encephalopathy) but acquired hyperbilirubinemia (T.Bil>5 mg/dl), pulmonary insufficiency and other complications at the same frequency as in the major Hx group. The survival rates in the three groups were 35.6%, 52.5%, and 48.6% at 3 years and 25.2%, 14.9%, and 24.3% at 5 years respectively. Curability rates (R0 to R1+2) were 76.7%, 54.5% and 50.0%, respectively. Preserving Hx tended to result in higher frequencies of positive transmural margins (e.g., cancer cells remaining around the right hepatic artery or the portal vein). CONCLUSIONS: Preserving hepatectomy for high-risk patients should be limited strictly to patients who do not have tumors which are not invading adjacent organs (e.g., T2) nor a segmental duct and are confined longitudinally to the right or the left.     

Pulse methylprednisolone with gammaglobulin as an initial treatment for acute Kawasaki disease

Approximately 15–20% of patients with Kawasaki disease (KD) are not responsive to high-dose intravenous gammaglobulin (IVIG). We have previously reported a predictive method for identifying IVIG-non-responsive patients (high-risk KD patients). We determined the safety and effectiveness of pulse methylprednisolone with high-dose IVIG (mPSL+IVIG) as a primary treatment for high-risk KD patients. Sixty-two high-risk KD patients were treated with pulse methylprednisolone 30 mg/kg over 2 h, followed by IVIG 2 g/kg over 24 h (mPSL+IVIG group) and were compared with a historical control group of 32 high-risk patients treated with IVIG 2 g/kg alone at the participating hospitals before this study was opened (IVIG group). High-risk patients were identified with at least two of three predictors (C-reactive protein ≥7 mg/dL, total bilirubin ≥0.9 mg/dL or aspartate aminotransferase ≥200 IU/L). Sixty-six percent (95% confidence interval [CI] 54–78%) of patients had a prompt defervescence in the mPSL+IVIG group compared with 44% (95% CI 26–62%) for the IVIG group (p = 0.048). Coronary artery lesions were observed in 24.2% (95% CI 13.2–35.2%) and 46.9% (95% CI 28.6–65.2%) of patients in the mPSL+IVIG and IVIG groups, respectively (p = 0.025). This is the first report showing that mPSL+IVIG is effective and safe as a primary treatment for high-risk KD patients.     

Minimum alveolar concentrations of sevoflurane for maintaining bispectral index below 50 in children

Objective: To determine minimum alveolar concentration (MAC) of sevoflurane for maintaining bispectral index (BIS) below 50 (MAC_BIS50) in children. Background: MAC_BIS50 of sevoflurane in adults was reported to be 0.97%, which has not been elucidated in children. Methods/Materials: Twenty children, American Society of Anesthesiologists physical status I or II, aged 1–8, were induced and anesthetized with sevoflurane in oxygen. After tracheal intubation, we started maintenance of anesthesia with endtidal sevoflurane concentrations of 2.6%. The endtidal sevoflurane concentration at which BIS was measured was predetermined by the up‐down method (with 0.2% as a step size). After 10 min at predetermined endtidal sevoflurane concentrations, BIS was measured for 1 min. MAC_BIS50 was determined using Dixon’s up‐down method and probit test. Result: MAC_BIS50 of sevoflurane was 2.83% (95% confidence intervals: 2.70–3.14) in children. Conclusions: MAC_BIS50 of sevoflurane in children was calculated to be three times as high as in adults. This indicates that high endtidal sevoflurane concentration is required to suppress electroencephalogram activity in children.