Nicotinic acid timed to feeding reverses tissue lipid accumulation and improves glucose control in obese Zucker rats[S].

Nicotinic acid (NiAc) is a potent inhibitor of lipolysis, acutely reducing plasma free fatty acid (FFA) concentrations. However, a major FFA rebound is seen during rapid NiAc washout, and sustained exposure is associated with tolerance development, with FFAs returning to pretreatment levels. Our aim was to find a rational NiAc dosing regimen that preserves FFA lowering, sufficient to reverse nonadipose tissue lipid accumulation and improve metabolic control, in obese Zucker rats. We compared feeding-period versus fasting-period NiAc dosing for 5 days: 12 h subcutaneous infusion (programmable, implantable mini-pumps) terminated by gradual withdrawal. It was found that NiAc timed to feeding decreased triglycerides in liver (-47%; P < 0.01) and heart (-38%; P < 0.05) and reduced plasma fructosamine versus vehicle. During oral glucose tolerance test, plasma FFA levels were reduced with amelioration of hyperglycemia and hypertriglyceridemia. Furthermore, timing NiAc to feeding resulted in a general downregulation of de novo lipogenesis (DNL) genes in liver. By contrast, NiAc timed to fasting did not reduce tissue lipids, ameliorate glucose intolerance or dyslipidemia, or alter hepatic DNL genes. In conclusion, NiAc dosing regimen has a major impact on metabolic control in obese Zucker rats. Specifically, a well-defined NiAc exposure, timed to feeding periods, profoundly improves the metabolic phenotype of this animal model.

Recombinant FVIII Products (Turoctocog Alfa and Turoctocog Alfa Pegol) Stable Up to 40°C.

PURPOSE: The stability under high-temperature conditions of factor VIII (FVIII) concentrates for replacement therapy is of critical importance to patients, particularly those who reside in, or travel to, regions with high ambient temperatures. Concerns about product stability may limit or prevent access to treatment for patients and may limit their ability to live a close-to-normal life. This study evaluated the effect of hot and humid storage conditions on the long-term stability of the recombinant FVIII products, turoctocog alfa and turoctocog alfa pegol. METHODS: Turoctocog alfa samples were assessed for stability at 30°C for 9 months or 40°C for 3 months following storage at 5°C for 21 or 27 months, respectively, while turoctocog alfa pegol samples were assessed at 30°C for 12 months or 40°C for 3 months following storage at 5°C for 18 or 27 months, respectively. In addition, turoctocog alfa and turoctocog alfa pegol dry powders were evaluated for stability at 5°C/ambient humidity (AH) for 30 months, 30°C/75% relative humidity (RH) for 12 months and 40°C/75% RH for 6 months. Both studies utilized a range of product strengths. Key stability assessments included oxidized forms, potency, water content and high molecular weight protein (HMWP). RESULTS: Both turoctocog alfa and turoctocog alfa pegol remained stable following storage at 40°C/75% RH for 3 months, and at single temperatures (5°C/AH, 30 and 40°C/75% RH), without any major increase in HMWP or any impairment of potency or water content. CONCLUSION: Turoctocog alfa and turoctocog alfa pegol offer stability at 40°C for up to 3 months without jeopardizing the quality of each product. These stability characteristics may offer patients flexibility with product storage and daily use.

Conjugated linoleic acid (CLA) inhibits expression of the Spot 14 (THRSP) and fatty acid synthase genes and impairs the growth of human breast cancer and liposarcoma cells.

Spot 14 (THRSP, S14) is a nuclear protein involved in the regulation of genes required for fatty acid synthesis in normal and malignant mammary epithelial and adipose cells. Harvatine and Bauman (1) reported that conjugated linoleic acid (CLA) inhibits S14 gene expression in bovine mammary and mouse adipose tissues and reduces milk fat production in cows. We hypothesized that CLA inhibits S14 gene expression in human breast cancer and liposarcoma cells and that this will retard their growth. Exposure of T47D breast cancer cells to a mixture of CLA isomers reduced the expression of the S14 and fatty acid synthase (FAS) genes. The mixture caused a dose-related inhibition of T47D cell growth, as did pure c9, t11 and t10, c12-CLA, but not linoleic acid. Similar effects were observed in MDA-MB-231 breast cancer cells. Provision of 8 mircoM palmitate fully (CLA mix, t10, c12-CLA) or partially (c9, t11-CLA) reversed the antiproliferative effect in T47D cells. CLA likewise suppressed levels of S14 and FAS mRNAs in liposarcoma cells and caused growth inhibition that was prevented by palmitic acid. CLA did not affect the growth of nonlipogenic HeLa cells or human fibroblasts. We conclude that as in bovine mammary and mouse adipose cells, CLA suppresses S14 and FAS gene expression in human breast cancer and liposarcoma cells. Rescue from the antiproliferative effect of CLA by palmitic acid indicates that reduced tumor lipogenesis is a major mechanism for the anticancer effects of CLA.

EndoS from Streptococcus pyogenes is hydrolyzed by the cysteine proteinase SpeB and requires glutamic acid 235 and tryptophans for IgG glycan-hydrolyzing activity.

BACKGROUND: The endoglycosidase EndoS and the cysteine proteinase SpeB from the human pathogen Streptococcus pyogenes are functionally related in that they both hydrolyze IgG leading to impairment of opsonizing antibodies and thus enhance bacterial survival in human blood. In this study, we further investigated the relationship between EndoS and SpeB by examining their in vitro temporal production and stability and activity of EndoS. Furthermore, theoretical structure modeling of EndoS combined with site-directed mutagenesis and chemical blocking of amino acids was used to identify amino acids required for the IgG glycan-hydrolyzing activity of EndoS. RESULTS: We could show that during growth in vitro S. pyogenes secretes the IgG glycan-hydrolyzing endoglycosidase EndoS prior to the cysteine proteinase SpeB. Upon maturation SpeB hydrolyzes EndoS that then loses its IgG glycan-hydrolyzing activity. Sequence analysis and structural homology modeling of EndoS provided a basis for further analysis of the prerequisites for IgG glycan-hydrolysis. Site-directed mutagenesis and chemical modification of amino acids revealed that glutamic acid 235 is an essential catalytic residue, and that tryptophan residues, but not the abundant lysine or the single cysteine residues, are important for EndoS activity. CONCLUSION: We present novel information about the amino acid requirements for IgG glycan-hydrolyzing activity of the immunomodulating enzyme EndoS. Furthermore, we show that the cysteine proteinase SpeB processes/degrades EndoS and thus emphasize the importance of the SpeB as a degrading/processing enzyme of proteins from the bacterium itself.

Vacancy ordering and superstructure formation in dry and hydrated strontium tantalate perovskites: a TEM perspective.

Crystal structures of Sr4(Sr2Ta2)O11 and Sr4(Sr1.92Ta2.08)O11.12, synthesized by solid state reaction technique in dry and hydrated state have been studied mainly using Transmission Electron Microscopy. Due to the lesser ability of X-rays to probe details in oxygen sublattice, the change in crystal symmetry due to ordering of oxygen vacancies could be detected better using Transmission Electron Microscopy. After detailed analysis through TEM, it was observed that no major change occurs in the cation sublattice. The TEM observations are compared with XRD data and discussed. The crystal symmetries and corresponding unit cells of all the perovskites based on the ordering of oxygen vacancies is deduced. Crystal unit cells based on the observations are proposed with ideal atomic coordinates. Finally an attempt is made to explain the water uptake behaviour of these perovskites based on the proposed crystal structure.

Fatty acid synthesis is a therapeutic target in human liposarcoma.

Liposarcomas (LS) are mesenchymal tumors that can recur after surgical resection and often do not respond to presently available medical therapies. This study demonstrates the dependence of LS on de novo long-chain fatty acid synthesis for growth. Lipogenesis can be impaired by inhibiting the activities of lipogenic enzymes, including acetyl CoA-carboxylase (ACC) and fatty acid synthase (FASN), or by suppressing the expression of key genes involved in the pathway and its regulation. The FASN inhibitors cerulenin and orlistat reduced the growth of two LS cell lines (LiSa2, SW872), as did inhibition of ACC with soraphen A. CDDO-Me, a synthetic triterpenoid, suppressed expression of Spot 14 and FASN genes and likewise inhibited LS cell growth. Importantly, the anti-proliferative effect of each agent was prevented by the co-administration of palmitate, the major product of cellular long-chain fatty acid synthesis. In stark contrast to LS cells, these compounds had no effect on the growth of fibroblasts. Four biochemically distinct agents that target critical points in the fatty acid synthetic pathway exert anti-proliferative effects on LS cells, and rescue of cell growth by palmitic acid suggests that reduced tumor cell lipogenesis mediates the growth inhibition. These findings warrant further studies aimed at the clinical exploitation of the dependence of LS cell growth on fatty acids.

Endoglycosidase treatment abrogates IgG arthritogenicity: importance of IgG glycosylation in arthritis.

The glycosylation status of IgG has been implicated in the pathology of rheumatoid arthritis. Earlier, we reported the identification of a novel secreted endo-beta-N-acetylglucosaminidase (EndoS), secreted by Streptococcus pyogenes that specifically hydrolyzes the beta-1,4-di-N-acetylchitobiose core of the asparagine-linked glycan of human IgG. Here, we analyzed the arthritogenicity of EndoS-treated collagen type II (CII)-specific mouse mAb in vivo. Endoglycosidase treatment of the antibodies inhibited the induction of arthritis in (BALB/c x B10.Q) F1 mice and induced a milder arthritis in B10.RIII mice as compared with the severe arthritis induced by non-treated antibodies. Furthermore, EndoS treatment did not affect the binding of IgG to CII and their ability to activate complement, but it resulted in reduced IgG binding to FcgammaR and disturbed the formation of stable immune complexes. Hence, the asparagine-linked glycan on IgG plays a crucial role in the development of arthritis.

Protein fibrils in nature can enhance amyloid protein A amyloidosis in mice: Cross-seeding as a disease mechanism.

Secondary, or amyloid protein A (AA), amyloidosis is a complication of chronic inflammatory diseases, both infectious and noninfectious. AA constitutes the insoluble fibrils, which are deposited in different organs, and is a major N-terminal part of the acute phase protein serum AA. It is not known why only some patients with chronic inflammation develop AA amyloidosis. Nucleation is a widely accepted mechanism in amyloidogenesis. Preformed amyloid-like fibrils act as nuclei in amyloid fibril formation in vitro, and AA amyloid fibrils and synthetic amyloid-like fibrils also may serve as seed for fibril formation in vivo. In addition to amyloid fibrils, there is a variety of similar nonmammalian protein fibrils with beta-pleated structure in nature. We studied three such naturally occurring protein fibrils: silk from Bombyx mori, Sup35 from Saccharomyces cerevisiae, and curli from Escherichia coli. Our results show that these protein fibrils exert amyloid-accelerating properties in the murine experimental AA amyloidosis, suggesting that such environment factors may be important risk factors in amyloidogenesis.

Identification of two protein-binding and functional regions of curli, a surface organelle and virulence determinant of Escherichia coli.

Curli are surface organelles of Escherichia coli. These fibrous proteins, formed by polymerization of a 15-kDa subunit, are expressed by E. coli strains associated with severe infections in humans. A remarkable property of curli is their ability to interact with a wide range of human proteins, interactions that contribute to the enhanced virulence of curli-expressing E. coli. To define the protein-binding region(s) of curli, we investigated the binding properties of overlapping synthetic peptides covering the curli subunit. Two peptides, one covering a 24-amino acid residue sequence in the NH(2)-terminal half of the subunit (NNS24) and one corresponding to the 26 COOH-terminal residues (VDQ26), were found to bind a number of human proteins. Physiochemical analysis revealed that NNS24 adopts a thermally stable beta-structure, and in solution the peptide forms soluble multimers, predominantly octamers. Intact curli are known to activate the proinflammatory and procoagulant contact system, and when added to human plasma, the NNS24 and VDQ26 peptides induced the release of the potent vasoactive peptide bradykinin. The results map important curli functions to the regions corresponding to the NNS24 and VDQ26 sequences.

EndoS and SpeB from Streptococcus pyogenes inhibit immunoglobulin-mediated opsonophagocytosis.

The human pathogen Streptococcus pyogenes primarily infects the upper respiratory tract and skin, but occasionally it disseminates and causes severe invasive disease with high mortality. This study revealed that the activity of extracellular EndoS, which hydrolyzes the functionally important N-linked oligosaccharides on opsonizing immunoglobulin G (IgG), contributes to increased survival of S. pyogenes in human blood ex vivo. The inability to kill the bacteria is due to reduced binding of IgG to Fc receptors and impaired classical pathway-mediated activation of complement. In addition, the activity of extracellular SpeB, which cleaves IgG into Fc and Fab fragments, also increases bacterial survival. This suggests that S. pyogenes expresses two enzymes, EndoS and SpeB, which modulate IgG by different mechanisms in order to evade the adaptive immune system.

Low antibody levels against cell wall-attached proteins of Streptococcus pyogenes predispose for severe invasive disease.

Acute-phase serum samples from 70 patients with group A streptococcal (GAS) invasive disease were analyzed for IgG antibodies against 6 recently characterized GAS virulence factors (SclA, SclB, GRAB, MtsA, EndoS, and IdeS) and SpeB. Antibody levels against the cell wall-attached GAS antigens SclA, SclB, and GRAB were significantly lower in patients with severe invasive disease (streptococcal toxic shock syndrome [STSS] and/or necrotizing fasciitis [NF]; n=35), compared with levels in patients with nonsevere GAS bacteremia (n=35). Among patients with severe invasive disease, significantly lower antibody levels against GRAB were found in patients with STSS (n=10) than in patients with NF (n=17). Antibody levels against SpeB in patients with severe bacteremia were similar to those in patients with nonsevere bacteremia, and levels in patients with STSS were similar to those in patients with NF. The data indicate that immunity to cell wall-attached proteins may play a role in the protection against severe invasive disease and that antibodies against GRAB may be of importance in the pathogenesis of STSS.