Bovine serum albumin displays luciferase-like activity in presence of luciferyl adenylate: Insights on the origin of protoluciferase activity and bioluminescence colours

Luciferyl adenylate, the key intermediate in beetle bioluminescence, is produced through adenylation of D-luciferin by beetle luciferases and also by mealworm luciferase-like enzymes which produce a weak red chemiluminescence. However, luciferyl adenylate is only weakly chemiluminescent in water at physiological pH and it is unclear how efficient bioluminescence evolved from its weak chemiluminescent properties. We found that bovine serum albumin (BSA) and neutral detergents enhance luciferyl adenylate chemiluminescence by three orders of magnitude, simulating the mealworm luciferase-like enzyme chemiluminescence properties. These results suggest that the beetle protoluciferase activity arose as an enhanced luciferyl adenylate chemiluminescence in the protein environment of the ancestral AMP-ligase. The predominance of luciferyl adenylate chemiluminescence in the red region under most conditions suggests that red luminescence is a more primitive condition that characterized the original stages of protobioluminescence, whereas yellow-green bioluminescence may have evolved later through the development of a more structured and hydrophobic active site. Copyright © 2006 John Wiley & Sons, Ltd.

Cryopreservation of dog spermatozoa: Effect of bovine serum albumin on acrosomal integrity and pregnancy rates after artificial insemination

The objective of the present study was to compare the in vitro and in vivo profile of frozen dog semen with Tris-bovine serum albumin (TB) and Tris-egg yolk (TE) extenders. Twenty dogs were used as donors. Each dog was stimulated by penile massage and only the sperm-rich fraction was collected weekly until 40 ejaculates were obtained. After macroscopic and microscopic analysis, equal parts of each ejaculate were diluted with TB and TE by the one-step method at 37 °C. The semen was added to 0.5-mL French straws which presented normal characteristics before freezing and after thawing. Acrosomal integrity was evaluated by double Trypan blue-Giemsa staining, in which alive intact (LI), alive reacted (LR), dead intact (DI) and dead reacted (DR) spermatozoa, were identified by the time of thawing and up to 4 h of incubation at 39 °C, the TE being significantly superior to TB (P<0,01) in the LI and LR variables. The TB being significantly superior to TE (P<0,01) in the DR variable. Female dogs in natural heat were submitted to artificial insemination, 20 receiving TE-semen and 20 receiving TB-semen with the Osiris probe (IMV, L'Aigle, France) and the numbers indicate that TE was significantly better than TB (P<0,01) to pregnancy rate and number of puppies/delivery. We concluded from this study, that TE was better than TB, because this, induced an eady acrossome reaction in dog's sperm.

Application of a serum/protein-free medium for the growth of mammalian cell lines and high level production of classical, variant and very virulent strain of infectious bursal disease virus (IBDV)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Sistema adesivo universal: influência da técnica de condicionamento e da pressão pulpar com soro fetal bovino ou água deionizada na resistência de união à dentina humana

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Oxygen tension affects histone remodeling of in vitro-produced embryos in a bovine model

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effect of pH, mucin and bovine serum on rifampicin permeability through Caco-2 cells

Rifampicin, a poorly soluble drug, has great importance in therapeutics as it is the main drug used to treat tuberculosis. The characterization of its permeability and the factors that influence it represent an important tool for predicting its bioavailability. Caco-2 cell monolayers were used as models of the intestinal mucosa to assess the uptake and transport of rifampicin and the effects of various experimental conditions were investigated, in order to establish the influence of these variables on rifampicin permeability. Different pHs (5.8, 6.8 and 7.4) in the apical medium, the presence or absence of mucin (3.0% w/v) in the donor site and the presence or absence of bovine serum albumin (4.0% v/v) in the receptor chamber were the evaluated conditions. The quantification of rifampicin in the apical or basolateral chambers was performed by a validated HPLC-UV method. The change in the donor chamber pH showed that permeability values were greater at pH 6.8, although this increase does not result in an alteration of the qualitative classification of rifampicin, which has high permeability. Mucin and bovine serum showed no effects on the permeability of rifampicin at the concentration tested. Overall, the current study suggests that pH, artificial mucin and bovine serum proteins have no influence on rifampicin permeability. Copyright (c) 2012 John Wiley & Sons, Ltd.

Endocannabinoid content in fetal bovine sera - unexpected effects on mononuclear cells and osteoclastogenesis

The major endocannabinoids (ECs) arachidonoylethanolamide (AEA) and 2-arachidonoylglycerol (2-AG) and related N-ethanolamines act as full and partial agonists at CB(1), CB(2), GPR55, PPAR and TRPV1 receptors to various degrees. These receptors are also expressed in immune cells like monocytes/macrophages where they regulate different cellular processes. In this study, potentially bioactive lipids in fetal bovine sera (FBS) were quantified by GC/MS. We found that several commercial FBS contain ECs and bioactive amounts of 2-AG (250-700 nM). We show that residual 2-AG from FBS can activate primary macrophages and increase migration and RANKL-stimulated osteoclastogenesis. Furthermore, 2-AG high-content sera specifically upregulated LPS-stimulated IL-6 expression in U937 cells. Polymyxin B beads may be used to selectively and efficiently remove 2-AG from sera, but not arachidonic acid and N-ethanolamines. In conclusion, 2-AG in cell culture media may significantly influence cellular experiments. CD14+ mononuclear cells which strongly express surface CB receptors may be particularly sensitive towards residual 2-AG from FBS. Therefore, the EC content in culture media should be controlled in biological experiments involving monocytes/macrophages.

Stable transduction of bovine TLR4 and bovine MD-2 into LPS-nonresponsive cells and soluble CD14 promote the ability to respond to LPS

The interaction of bovine cells with lipopolysaccharide (LPS) was explored using human embryo kidney (HEK) 293 cell line stably transduced with bovine toll-like receptor-4 (TLR4) alone or in combination with bovine MD-2. These lines and mock-transduced HEK293 cells were tested by flow cytometry for LPS-fluorescein isothiocyanate (LPS-FITC) binding, nuclear factor kappa B (NFkappaB) activation, interleukin-8 (IL-8) production and interferon-beta mRNA expression/interferon (IFN) type I production. Whereas bovine TLR4 was sufficient to promote binding of high concentrations of LPS-FITC, both bovine TLR4 and MD-2 were required for activation by LPS, as assessed by NFkappaB activation and IL-8 production. Induction of IFN bioactivity was not observed in doubly transduced HEK293 cells, and no evidence for IFN-beta mRNA induction in response to LPS was obtained, although cells responded by IFN-beta mRNA expression to stimulation by Sendai virus and poly-inosinic acid-poly-cytidylic acid (poly(I:C)). Cells stably transduced with both bovine TLR4 and bovine MD-2 responded to LPS by IL-8 production, in decreasing order, in the presence of fetal bovine serum (FCS), of human serum, and of human serum albumin (HSA). The reduced activity in the presence of HSA could be restored by the addition of soluble CD14 (sCD14) but not of LPS binding protein (LBP). This is in contrast to macrophages which show a superior response to LPS in the presence of HSA when compared with macrophages stimulated by LPS in the presence of FCS. This suggests that macrophages but not HEK293 cells express factors rendering LPS stimulation serum-independent. Stably double-transduced cells reacted, in decreasing order, to LPS from Rhodobacter sphaeroides, to LPS from Escherichia coli, to synthetic lipd-IVa (compound 406), to diphosphoryl-lipid-A (S. minnesota) and to monophosphoryl-lipid-A (S. minnesota). They failed to react to the murine MD-2/TLR4 ligand taxol. This resembles the reactivity of bovine macrophages with regard to sensitivity (ED(50)) and order of potency but is distinct from the reactivity pattern of other species. This formally establishes that in order to react to LPS, cattle cells require serum factors (e.g. sCD14) and cell-expressed factors such as MD-2 and TLR4. The cell lines described are the first of a series expressing defined pattern recognition receptors (PRR) of bovine origin. They will be useful in the study of the interaction of the bovine TLR4-MD-2 complex and Gram-negative bovine pathogens, e.g. the agents causing Gram-negative bovine mastitis.

Generation of long, fully modified, and serum-resistant oligonucleotides by rolling circle amplification

Rolling Circle Amplification (RCA) is an isothermal enzymatic method generating single-stranded DNA products consisting of concatemers containing multiple copies of the reverse complement of the circular template precursor. Little is known on the compatibility of modified nucleoside triphosphates (dN*TPs) with RCA, which would enable the synthesis of long, fully modified ssDNA sequences. Here, dNTPs modified at any position of the scaffold were shown to be compatible with rolling circle amplification, yielding long (>1 kb), and fully modified single-stranded DNA products. This methodology was applied for the generation of long, cytosine-rich synthetic mimics of telomeric DNA. The resulting modified oligo-nucleotides displayed an improved resistance to fetal bovine serum.

Photochemical protease: Site-specific photocleavage of hen egg lysozyme and bovine serum albumin

Site-specific photocleavage of hen egg lysozyme and bovine serum albumin (BSA) by N-(l-phenylalanine)-4-(1-pyrene)butyramide (Py-Phe) is reported. Py-Phe binds to lysozyme and BSA with binding constants 2.2 ± 0.3 × 105 M−1 and 6.5 ± 0.4 × 107 M−1, respectively. Photocleavage of lysozyme and BSA was achieved with high specificity when a mixture of protein, Py-Phe, and an electron acceptor, cobalt(III) hexammine (CoHA), was irradiated at 344 nm. Quantum yields of photocleavage of lysozyme and BSA were 0.26 and 0.0021, respectively. No protein cleavage was observed in the absence of Py-Phe, CoHA, or light. N-terminal sequencing of the protein fragments indicated a single cleavage site of lysozyme between Trp-108 and Val-109, whereas the cleavage of BSA was found to be between Leu-346 and Arg-347. Laser flash photolysis studies of a mixture of protein, Py-Phe, and CoHA showed a strong transient with absorption centered at ≈460 nm, corresponding to pyrene cation radical. Quenching of the singlet excited state of Py-Phe by CoHA followed by the reaction of the resulting pyrenyl cation radical with the protein backbone may be responsible for the protein cleavage. The high specificity of photocleavage may be valuable in targeting specific sites of proteins with small molecules.

Generation and maturation of dendritic cells for clinical application under serum-free conditions

Monocyte-derived dendritic cells (MoDCs) in clinical use for cancer immunotherapy are ideally generated in serum-free medium (SFM) with inclusion of a suitable maturation factor toward the end of the incubation period. Three good manfacturing practice (GMP) grade SFMs (AIM-V, X-VIVO 15, and X-VIVO 20) were compared with RPMI-1640, supplemented with 10% fetal bovine serum or 10% human serum. DCs generated for 7 days in SFM were less mature and secreted less interleukin (IL) 12p70 and IL-10 than DCs generated in 10% serum. DC yield was comparable in SFMs, and a greater proportion of cells was viable after maturation. Toll-like receptor (TLR) ligands were compared for their ability to induce cytokine secretion under serum-free conditions in the presence of interferon (IFN) gamma. With the exception of Poly I:C, TLR ligands stimulated high levels of IL-10 secretion. High levels of IL-12p70 were induced by two TLR4-mediated stimuli, lipopolysaccharide and Ribomunyl, a clinical-grade bacterial extract. When T-cell responses were compared in allogeneic mixed leukocyte reaction, DCs stimulated with Ribomunyl induced higher levels of IFN gamma than DCs stimulated with the cytokine cocktail: tumor necrosis factor-alpha, IL-1 beta, IL-6, and prostaglandin E-2. In the presence of IL-10 neutralizing antibodies, DC IL-12p70 production and T-cell IFN gamma were increased in vitro. Similarly, DCs stimulated with Ribomunyl, IFN gamma, and anti-IL-10 induced high levels of tetanus toxoid-specific T-cell proliferation and IFN gamma secretion. Thus, MoDCs generated ill SFM efficiently stimulate T-cell IFN gamma production after maturation in the presence of a clinical-grade TLR4 agonist and IL-10 neutralization.

Serum-free process development:improving the yield and consistency of human mesenchymal stromal cell production

Background aims: The cost-effective production of human mesenchymal stromal cells (hMSCs) for off-the-shelf and patient specific therapies will require an increasing focus on improving product yield and driving manufacturing consistency. Methods: Bone marrow-derived hMSCs (BM-hMSCs) from two donors were expanded for 36 days in monolayer with medium supplemented with either fetal bovine serum (FBS) or PRIME-XV serum-free medium (SFM). Cells were assessed throughout culture for proliferation, mean cell diameter, colony-forming potential, osteogenic potential, gene expression and metabolites. Results: Expansion of BM-hMSCs in PRIME-XV SFM resulted in a significantly higher growth rate (P < 0.001) and increased consistency between donors compared with FBS-based culture. FBS-based culture showed an inter-batch production range of 0.9 and 5 days per dose compared with 0.5 and 0.6 days in SFM for each BM-hMSC donor line. The consistency between donors was also improved by the use of PRIME-XV SFM, with a production range of 0.9 days compared with 19.4 days in FBS-based culture. Mean cell diameter has also been demonstrated as a process metric for BM-hMSC growth rate and senescence through a correlation (R² = 0.8705) across all conditions. PRIME-XV SFM has also shown increased consistency in BM-hMSC characteristics such as per cell metabolite utilization, in vitro colony-forming potential and osteogenic potential despite the higher number of population doublings. Conclusions: We have increased the yield and consistency of BM-hMSC expansion between donors, demonstrating a level of control over the product, which has the potential to increase the cost-effectiveness and reduce the risk in these manufacturing processes.

Bovine serum albumin as the dominant form of dietary protein reduces subcutaneous fat mass, plasma leptin and plasma corticosterone in high fat-fed C57/BL6J mice

Acknowledgements The authors thank Harriett Schellekens from the University College Cork and Paula O’Connor from Teagasc Moorepark Food Research Centre for their assistance in procuring laboratory space and equipment. The present study was funded by Teagasc. B. L. M. was funded by the Walsh Fellowship Program. J. R. S. was supported by a 1000-talents professorship from the Chinese government. The funding bodies had no input on the design of the study or in the interpretation of the data.

Cationic Bovine Serum Albumin (CBA) conjugated Poly Lactic-co-Glycolic Acid (PLGA) nanoparticles for extended delivery of methotrexate into brain tumor

Current treatment strategies for the treatment of brain tumor have been hindered primarily by the presence of highly lipophilic insurmountable blood-brain barrier (BBB). The purpose of current research was to investigate the efficiency of engineered biocompatible polymeric nanoparticles (NPs) as drug delivery vehicle to bypass the BBB and enhance biopharmaceutical attributes of anti-metabolite methotrexate (MTX) encapsulated NPs. The NPs were prepared by solvent diffusion method using cationic bovine serum albumin (CBA), and characterized for physicochemical parameters such as particle size, polydispersity index, and zeta-potential; while the surface modification was confirmed by FTIR, and NMR spectroscopy. Developed NPs exhibited zestful relocation of FITC tagged NPs across BBB in albino rats. Further, hemolytic studies confirmed them to be non-toxic and biocompatible as compared to free MTX. In vitro cytotoxicity assay of our engineered NPs on HNGC1 tumor cells proved superior uptake in tumor cells; and elicited potent cytotoxic effect as compared to plain NPs and free MTX solution. The outcomes of the study evidently indicate the prospective of CBA conjugated poly (D,L-lactide-co-glycolide) (PLGA) NPs loaded with MTX in brain cancer bomber with amplified capability to circumvent BBB.