84 research outputs found
Interactions between cytosolic components of the NADPH oxidase: p40phox interacts with both p67phox and p47phox
Dual specificity antibodies using a double-stranded oligonucleotide bridge
AbstractThe covalent conjugation of oligonucleotides to antibody Fab’ fragments was optimized by using oligonucleotides modified with a hexaethylene linker arm bearing three amino groups. One oligonucleotide was coupled to antibody of one specificity and a complementary oligonucleotide to antibody of a second specificity. The antibodies were then allowed to hybridize by base pairing of the complementary nucleotide sequences and the generation of bispecific antibody was analyzed on SDS-PAGE and confirmed using BIAcore analysis. The strategy of complementary oligonucleotide-linked bispecific molecules is not limited to antibodies but is applicable to linking any two molecules of different characteristics
Industrial derivative of tallow: a promising renewable substrate for microbial lipid, single-cell protein and lipase production by Yarrowia lipolytica
The aim of the present study was to assess the potential of
valorisation of a solid industrial derivative of tallow, composed of
saturated free-fatty acids ("stearin"), by fermentations carried out by
the yeast Yarrowia lipolytica ACA-DC 50109 in order to produce
microbial lipid, biomass and extra-cellular lipase. High quantities of
biomass were produced (biomass yield of around 1.1 \ub1 0.1 g of
total biomass produced per g of fat consumed) when the organism was
grown in shake flasks, regardless of the concentration of
extra-cellular nitrogen present. Cellular lipids accumulated in notable
quantities regardless of the nitrogen availability of the medium,
though this process was clearly favoured at high initial fat and low
initial nitrogen concentrations. The maximum quantity of fat produced
was 7.9 mg/ml corresponding to 52.0% (wt/wt) of lipid in the dry
biomass. Lipase production was critically affected by the medium
composition and its concentration clearly increased with increasing
concentrations of fat and extra-cellular nitrogen concentration
reaching a maximum level of 2.50 IU/ml. Lipase concentration decreased
in the stationary growth phase. In bioreactor trials, in which higher
agitation and aeration conditions were employed compared with the
equivalent trial in the flasks, significantly higher quantities of
biomass were produced (maximum concentration 30.5 mg/ml, yield of 1.6 g
of total biomass produced per g of fat consumed) while remarkably lower
quantities of cellular lipids and extra-cellular lipase were
synthesised. Numerical models successfully simulated both conversion of
substrate fat into biomass and production and subsequent hydrolysis of
extra-cellular lipase and presented a satisfactory predictive ability
verifying the potential for single-cell protein and lipase production
by Yarrowia lipolytica ACA-DC 50109. In all cultures, the mycelial form
of the culture was dominant with few single cells present
Industrial derivative of tallow: a promising renewable substrate for microbial lipid, single-cell protein and lipase production by Yarrowia lipolytica
New Insights for RANKL as a Proinflammatory Modulator in Modeled Inflammatory Arthritis
Receptor activator of nuclear factor-κB ligand (RANKL), a member of the Tumor Necrosis Factor (TNF) superfamily, constitutes the master regulator of osteoclast formation and bone resorption, whereas its involvement in inflammatory diseases remains unclear. Here, we used the human TNF transgenic mouse model of erosive inflammatory arthritis to determine if the progression of inflammation is affected by either genetic inactivation or overexpression of RANKL in transgenic mouse models. TNF-mediated inflammatory arthritis was significantly attenuated in the absence of functional RANKL. Notably, TNF overexpression could not compensate for RANKL-mediated osteopetrosis, but promoted osteoclastogenesis between the pannus and bone interface, suggesting RANKL-independent mechanisms of osteoclastogenesis in inflamed joints. On the other hand, simultaneous overexpression of RANKL and TNF in double transgenic mice accelerated disease onset and led to severe arthritis characterized by significantly elevated clinical and histological scores as shown by aggressive pannus formation, extended bone resorption, and massive accumulation of inflammatory cells, mainly of myeloid origin. RANKL and TNF cooperated not only in local bone loss identified in the inflamed calcaneous bone, but also systemically in distal femurs as shown by microCT analysis. Proteomic analysis in inflamed ankles from double transgenic mice overexpressing human TNF and RANKL showed an abundance of proteins involved in osteoclastogenesis, pro-inflammatory processes, gene expression regulation, and cell proliferation, while proteins participating in basic metabolic processes were downregulated compared to TNF and RANKL single transgenic mice. Collectively, these results suggest that RANKL modulates modeled inflammatory arthritis not only as a mediator of osteoclastogenesis and bone resorption but also as a disease modifier affecting inflammation and immune activation
Direct measurement of the substrate preference of uracil-DNA glycosylase
Site-directed mutants of the herpes simplex virus type 1 uracil-DNA glycosylase lacking catalytic activity have been used to probe the substrate recognition of this highly conserved and ubiquitous class of DNA-repair enzyme utilizing surface plasmon resonance. The residues aspartic acid-88 and histidine-210, implicated in the catalytic mechanism of the enzyme (Savva, R., McAuley-Hecht, K., Brown, T., and Pearl, L. (1995) Nature 373, 487–493; Slupphaug, G., Mol, C. D., Kavli, B., Arvai, A. S., Krokan, H. E. and Tainer, J. A. (1996) Nature 384, 87–92) were separately mutated to asparagine to allow investigations of substrate recognition in the absence of catalysis. The mutants were shown to be correctly folded and to lack catalytic activity. Binding to single- and double-stranded oligonucleotides, with or without uracil, was monitored by real-time biomolecular interaction analysis using surface plasmon resonance. Both mutants exhibited comparable rates of binding and dissociation on the same uracil-containing substrates. Interaction with single-stranded uracil-DNA was found to be stronger than with double-stranded uracil-DNA, and the binding to Gua:Ura mismatches was significantly stronger than that to Ade:Ura base pairs suggesting that the stability of the base pair determines the efficiency of interaction. Also, there was negligible interaction between the mutants and single- or double-stranded DNA lacking uracil, or with DNA containing abasic sites. These results suggest that it is uracil in the DNA, rather than DNA itself, that is recognized by the uracil-DNA glycosylases
Differential detection of nuclear envelope autoantibodies in primary biliary cirrhosis using routine and alternative methods
Complexity Theory for a New Managerial Paradigm: A Research Framework
In this work, we supply a theoretical framework of how organizations
can embed complexity management and sustainable development into their policies
and actions. The proposed framework may lead to a new management paradigm,
attempting to link the main concepts of complexity theory, change management,
knowledge management, sustainable development, and cybernetics. We highlight
how the processes of organizational change have occurred as a result of the move to
adapt to the changes in the various global and international business environments
and how this transformation has led to the shift toward the present innovation
economy. We also point how organizational change needs to deal with sustainability,
so that the change may be consistent with present needs, without compromising
the future
Characterization of Flavonoid Subgroups and Hydroxy Substitution by HPLC-MS/MS
Abstract: HPLC-DAD coupled with mass spectrometry in the positive ionization mode was applied to study the fragmentation of twelve selected flavonoids. Compounds belonging to all the major subgroups found in common plants, i.e. flavonols, flavones, dihydroflavonols, flavanones and flavanols were studied. Compound standards were injected into the spectrometer and produced characteristic mass spectra. The fragmentation of each compound was studied and it was shown that the dehydration and carbon monoxide losses from the [M+H] + ion by the members of each subgroup produced specific fragments, thus allowing the characterization of the flavonoid subgroups. Moreover, fragments resulting from fission of the C-rings are specific of each subgroup and revealed the substitution pattern of A- and B-rings. In order to verify the identifying efficiency of the positive ionization mode through these characteristic fragmentations, the unknown flavonoids of an Origanum vulgare diethyl ether extract were separated with the HPLC system and the major peaks were successfully identified with the mass spectrometer
Allotypic variation in antigen processing controls antigenic peptide generation from SARS-CoV-2 S1 spike glycoprotein
Population genetic variability in immune system genes can often underlie
variability in immune responses to pathogens. Cytotoxic T-lymphocytes
are emerging as critical determinants of both severe acute respiratory
syndrome coronavirus 2 infection severity and long-term immunity, after
either recovery or vaccination. A hallmark of coronavirus disease 2019
is its highly variable severity and breadth of immune responses between
individuals. To address the underlying mechanisms behind this
phenomenon, we analyzed the proteolytic processing of S1 spike
glycoprotein precursor antigenic peptides across ten common allotypes of
endoplasmic reticulum aminopeptidase 1 (ERAP1), a polymorphic
intracellular enzyme that can regulate cytotoxic T-lymphocyte responses
by generating or destroying antigenic peptides. We utilized a systematic
proteomic approach that allows the concurrent analysis of hundreds of
trimming reactions in parallel, thus better emulating antigen processing
in the cell. While all ERAP1 allotypes were capable of producing optimal
ligands for major histocompatibility complex class I molecules,
including known severe acute respiratory syndrome coronavirus 2
epitopes, they presented significant differences in peptide sequences
produced, suggesting allotype-dependent sequence biases. Allotype 10,
previously suggested to be enzymatically deficient, was rather found to
be functionally distinct from other allotypes. Our findings suggest that
common ERAP1 allotypes can be a major source of heterogeneity in antigen
processing and through this mechanism contribute to variable immune
responses in coronavirus disease 2019
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