Adeno-associated virus-8-Mediated intravenous transfer of myostatin propeptide leads to systemic functional improvements of slow but not fast muscle

Myostatin is a member of the transformating growth factor-_ (TGF-_) superfamily of proteins and is produced almost exclusively in skeletal muscle tissue, where it is secreted and circulates as a serum protein. Myostatin acts as a negative regulator of muscle mass through the canonical SMAD2/3/4 signaling pathway. Naturally occurring myostatin mutants exhibit a ‘double muscling’ phenotype in which muscle mass is dramatically increased as a result of both hypertrophy and hyperplasia. Myostatin is naturally inhibited by its own propeptide; therefore, we assessed the impact of adeno associated virus-8 (AAV8) myostatin propeptide vectors when systemically introduced in MF-1 mice. We noted a significant systemic increase in muscle mass in both slow and fast muscle phenotypes, with no evidence of hyperplasia; however, the nuclei-to- cytoplasm ratio in all myofiber types was significantly reduced. An increase in muscle mass in slow (soleus) muscle led to an increase in force output; however, an increase in fast (extensor digitorum longus [EDL]) muscle mass did not increase force output. These results suggest that the use of gene therapeutic regimens of myostatin inhibition for age-related or disease-related muscle loss may have muscle-specific effects

Comparative analysis of involvement of UGT1 and UGT2 splice variants of UDP-galactose transporter in glycosylation of macromolecules in MDCK and CHO cell lines

Nucleotide sugar transporters deliver nucleotide sugars into the Golgi apparatus and endoplasmic reticulum. This study aimed to further characterize mammalian UDP-galactose transporter (UGT) in MDCK and CHO cell lines. MDCK-RCAr and CHO-Lec8 mutant cell lines are defective in UGT transporter, although they exhibit some level of galactosylation. Previously, only single forms of UGT were identified in both cell lines, UGT1 in MDCK cells and UGT2 in CHO cells. We have identified the second UGT splice variants in CHO (UGT1) and MDCK (UGT2) cells. Compared to UGT1, UGT2 is more abundant in nearly all examined mammalian tissues and cell lines, but MDCK cells exhibit different relative distribution of both splice variants. Complementation analysis demonstrated that both UGT splice variants are necessary for N- and O-glycosylation of proteins. Both mutant cell lines produce chondroitin-4-sulfate at only a slightly lower level compared to wild-type cells. This defect is corrected by overexpression of both UGT splice variants. MDCK-RCAr mutant cells do not produce keratan sulfate and this effect is not corrected by either UGT splice variant, overexpressed either singly or in combination. Here we demonstrate that both UGT splice variants are important for glycosylation of proteins. In contrast to MDCK cells, MDCK-RCAr mutant cells may possess an additional defect within the keratan sulfate biosynthesis pathway

Endometrial stromal sarcoma: a population-based analysis

To determine independent prognostic factors for the survival of patients with endometrial stromal sarcoma (ESS), data were abstracted from the Surveillance, Epidemiology, and End Results (SEER) database of the National Cancer Institute from 1988 to 2003. Kaplan–Meier and Cox proportional hazards models were used for analyses. Of 831 women diagnosed with ESS, the median age was 52 years (range: 17–96 years). In total, 59.9% had stage I, 5.1% stage II, 14.9% stage III, and 20.1% had stage IV disease. Overall, 13.0, 36.1, and 34.7% presented with grades 1, 2, and 3, respectively. Patients with stage I–II vs III–IV disease had 5 years DSS of 89.3% vs 50.3% (P<0.001) and those with grades 1, 2, and 3 cancers had survivals of 91.4, 95.4, and 42.1% (P<0.001). In multivariate analysis, older patients, black race, advanced stage, higher grade, lack of primary surgery, and nodal metastasis were independent prognostic factors for poorer survival. In younger women (<50 years) with stage I–II disease, ovarian-sparing procedures did not adversely impact survival (91.9 vs 96.2%; P=0.1). Age, race, primary surgery, stage, and grade are important prognostic factors for ESS. Excellent survival in patients with grade 1 and 2 disease of all stages supports the concept that these tumors are significantly different from grade 3 tumors. Ovarian-sparing surgeries may be considered in younger patients with early-stage disease

Tyrosine Sulfation of Native Mouse Psgl-1 Is Required for Optimal Leukocyte Rolling on P-Selectin In Vivo

We recently demonstrated that tyrosine sulfation is an important contributor to monocyte recruitment and retention in a mouse model of atherosclerosis. P-selectin glycoprotein ligand-1 (Psgl-1) is tyrosine-sulfated in mouse monocyte/macrophages and its interaction with P-selectin is important in monocyte recruitment in atherosclerosis. However, whether tyrosine sulfation is required for the P-selectin binding function of mouse Psgl-1 is unknown. Here we test the function of native Psgl-1 expressed in leukocytes lacking endogenous tyrosylprotein sulfotransferase (TPST) activity.Psgl-1 function was assessed by examining P-selectin dependent leukocyte rolling in post-capillary venules of C57BL6 mice transplanted with hematopoietic progenitors from wild type (WT → B6) or Tpst1;Tpst2 double knockout mice (Tpst DKO → B6) which lack TPST activity. We observed that rolling flux fractions were lower and leukocyte rolling velocities were higher in Tpst DKO → B6 venules compared to WT → B6 venules. Similar results were observed on immobilized P-selectin in vitro. Finally, Tpst DKO leukocytes bound less P-selectin than wild type leukocytes despite equivalent surface expression of Psgl-1.These findings provide direct and convincing evidence that tyrosine sulfation is required for optimal function of mouse Psgl-1 in vivo and suggests that tyrosine sulfation of Psgl-1 contributes to the development of atherosclerosis

Efficacy of a Non-Hypercalcemic Vitamin-D2 Derived Anti-Cancer Agent (MT19c) and Inhibition of Fatty Acid Synthesis in an Ovarian Cancer Xenograft Model

BACKGROUND:Numerous vitamin-D analogs exhibited poor response rates, high systemic toxicities and hypercalcemia in human trials to treat cancer. We identified the first non-hypercalcemic anti-cancer vitamin D analog MT19c by altering the A-ring of ergocalciferol. This study describes the therapeutic efficacy and mechanism of action of MT19c in both in vitro and in vivo models. METHODOLOGY/PRINCIPAL FINDING:Antitumor efficacy of MT19c was evaluated in ovarian cancer cell (SKOV-3) xenografts in nude mice and a syngenic rat ovarian cancer model. Serum calcium levels of MT19c or calcitriol treated animals were measured. In-silico molecular docking simulation and a cell based VDR reporter assay revealed MT19c-VDR interaction. Genomewide mRNA analysis of MT19c treated tumors identified drug targets which were verified by immunoblotting and microscopy. Quantification of cellular malonyl CoA was carried out by HPLC-MS. A binding study with PPAR-Y receptor was performed. MT19c reduced ovarian cancer growth in xenograft and syngeneic animal models without causing hypercalcemia or acute toxicity. MT19c is a weak vitamin-D receptor (VDR) antagonist that disrupted the interaction between VDR and coactivator SRC2-3. Genome-wide mRNA analysis and western blot and microscopy of MT19c treated xenograft tumors showed inhibition of fatty acid synthase (FASN) activity. MT19c reduced cellular levels of malonyl CoA in SKOV-3 cells and inhibited EGFR/phosphoinositol-3kinase (PI-3K) activity independently of PPAR-gamma protein. SIGNIFICANCE:Antitumor effects of non-hypercalcemic agent MT19c provide a new approach to the design of vitamin-D based anticancer molecules and a rationale for developing MT19c as a therapeutic agent for malignant ovarian tumors by targeting oncogenic de novo lipogenesis

The Staphylococcus aureus superantigen SElX is a bifunctional toxin that inhibits neutrophil function:SElX Inhibits Neutrophil Function

Bacterial superantigens (SAgs) cause Vβ-dependent T-cell proliferation leading to immune dysregulation associated with the pathogenesis of life-threatening infections such as toxic shock syndrome, and necrotizing pneumonia. Previously, we demonstrated that staphylococcal enterotoxin-like toxin X (SElX) from Staphylococcus aureus is a classical superantigen that exhibits T-cell activation in a Vβ-specific manner, and contributes to the pathogenesis of necrotizing pneumonia. Here, we discovered that SElX can also bind to neutrophils from human and other mammalian species and disrupt IgG-mediated phagocytosis. Site-directed mutagenesis of the conserved sialic acid-binding motif of SElX abolished neutrophil binding and phagocytic killing, and revealed multiple glycosylated neutrophil receptors for SElX binding. Furthermore, the neutrophil binding-deficient mutant of SElX retained its capacity for T-cell activation demonstrating that SElX exhibits mechanistically independent activities on distinct cell populations associated with acquired and innate immunity, respectively. Finally, we demonstrated that the neutrophil-binding activity rather than superantigenicity is responsible for the SElX-dependent virulence observed in a necrotizing pneumonia rabbit model of infection. Taken together, we report the first example of a SAg, that can manipulate both the innate and adaptive arms of the human immune system during S. aureus pathogenesis