86 research outputs found
Profiling the Hydrolysis of Isolated Grape Berry Skin Cell Walls by Purified Enzymes
The
unraveling of crushed grapes by maceration enzymes during winemaking
is difficult to study because of the complex and rather undefined
nature of both the substrate and the enzyme preparations. In this
study we simplified both the substrate, by using isolated grape skin
cell walls, and the enzyme preparations, by using purified enzymes
in buffered conditions, to carefully follow the impact of the individual
and combined enzymes on the grape skin cell walls. By using cell wall
profiling techniques we could monitor the compositional changes in
the grape cell wall polymers due to enzyme activity. Extensive enzymatic
hydrolysis, achieved with a preparation of pectinases or pectinases
combined with cellulase or hemicellulase enzymes, completely removed
or drastically reduced levels of pectin polymers, whereas less extensive
hydrolysis only opened up the cell wall structure and allowed extraction
of polymers from within the cell wall layers. Synergistic enzyme activity
was detectable as well as indications of specific cell wall polymer
associations
Following the Compositional Changes of Fresh Grape Skin Cell Walls during the Fermentation Process in the Presence and Absence of Maceration Enzymes
Cell
wall profiling technologies were used to follow compositional
changes that occurred in the skins of grape berries (from two different
ripeness levels) during fermentation and enzyme maceration. Multivariate
data analysis showed that the fermentation process yielded cell walls
enriched in hemicellulose components because pectin was solubilized
(and removed) with a reduction as well as exposure of cell wall proteins
usually embedded within the cell wall structure. The addition of enzymes
caused even more depectination, and the enzymes unravelled the cell
walls enabling better access to, and extraction of, all cell wall
polymers. Overripe grapes had cell walls that were extensively hydrolyzed
and depolymerized, probably by natural grape-tissue-ripening enzymes,
and this enhanced the impact that the maceration enzymes had on the
cell wall monosaccharide profile. The combination of the techniques
that were used is an effective direct measurement of the hydrolysis
actions of maceration enzymes on the cell walls of grape berry skin
An HIV-1 Envelope Glycoprotein Trimer with an Embedded IL-21 Domain Activates Human B Cells
<div><p>Broadly neutralizing antibodies (bNAbs) that target the HIV-1 envelope glycoproteins (Env) can prevent virus acquisition, but several Env properties limit its ability to induce an antibody response that is of sufficient quantity and quality. The immunogenicity of Env can be increased by fusion to co-stimulatory molecules and here we describe novel soluble Env trimers with embedded interleukin-4 (IL-4) or interleukin-21 (IL-21) domains, designed to activate B cells that recognize Env. In particular, the chimeric Env<sub>IL-21</sub> molecule activated B cells efficiently and induced the differentiation of antibody secreting plasmablast-like cells. We studied whether we could increase the activity of the embedded IL-21 by designing a chimeric IL-21/IL-4 (ChimIL-21/4) molecule and by introducing amino acid substitutions in the receptor binding domain of IL-21 that were predicted to enhance its binding. In addition, we incorporated IL-21 into a cleavable Env trimer and found that insertion of IL-21 did not impair Env cleavage, while Env cleavage did not impair IL-21 activity. These studies should guide the further design of chimeric proteins and Env<sub>IL-21</sub> may prove useful in improving antibody responses against HIV-1.</p></div
Immunoglobulin production by B cells stimulated with Env<sub>wt</sub>, Env<sub>IL-4</sub> and Env<sub>IL-21</sub> molecules and controls.
<p>IgG, IgA and IgM levels secreted by the B cells from human PBMCs cultured with (A) CD40L/IL-10 and (B) CD40L/IL-4/IL-10. Data are representative of three independent experiments showing similar results. Immunoglobulin secretion by B cells from different donors cultured with Env<sub>wt</sub> and Env<sub>IL-21</sub> molecules in (C) CD40L/IL-10 and (D) CD40L/IL-4/IL-10 milieu. Culture supernatant from mock transfected 293T cells was used as a negative control and the values were deducted from the test values. Data represent the fold change values compared to Env<sub>wt</sub> from at least 12 donors and each donor sample was tested in duplicate. (E) The expression of cell surface markers CD38 and CD27 on B cells cultured with Env<sub>wt</sub>, Env<sub>IL-21,</sub> Env<sub>ChimIL-21/4</sub> supernatants and controls in CD40L/IL-10 milieu. Data are representative of three experiments using B cells from three different donors. (F) The expression of CD38 cell surface marker treated with different Env<sub>IL-21</sub> constructs and controls in CD40L/IL-10 milieu. Data are representative of six experiments using B cells from six different donors.</p
Schematic and expression of the cleavable Env<sub>IL-21</sub>.
<p>Linear (A) and cartoon (B) representation of the Env<sub>IL-21</sub> and clEnv<sub>IL-21</sub> proteins. Cleaved proteins were created by introducing a stop codon in front of the isoleucine zipper (IZ) trimerization domain in the Env<sub>IL-21</sub>. (C) SDS-PAGE analysis of chimeric uncleaved and cleaved Env<sub>IL-21</sub> constructs.</p
Immunoglobulin secretion from B cells cultured with Env<sub>wt</sub>, Env<sub>IL-21</sub>, clEnv and clEnv<sub>IL-21</sub> molecules in the presence of (A) CD40L/IL-10 and (B) CD40L/IL-4/IL-10.
<p>Data are representative of two independent experiments using B cells from two different donor, each tested in duplicate. (C) The expression of cell surface markers CD38 and CD27 on B cells cultured with Env<sub>wt</sub>, Env<sub>IL-21</sub>, clEnv and clEnv<sub>IL-21</sub>supernatants in medium supplemented with CD40L/IL-10. Data are representative of three independent experiments using B cells from three donors. (D) The expression of CD38 cell surface marker treated with different cleaved Env (clEnv) and cleaved Env<sub>IL-21</sub> (clEnv<sub>IL-21</sub>) constructs in CD40L/IL-10 milieu. Data are representative of six experiments using B cells from six donors.</p
Antigenic characterization of Env<sub>IL-4</sub> and Env<sub>IL-21</sub> molecules.
<p>ELISA reactivity of Env<sub>IL-4</sub> and Env<sub>IL-21</sub> with 2G12 and HIV-Ig (A); b12 and CD4-IgG2 (B); and 48d (CD4i) in the absence and presence of sCD4 at 1 µg/ml (C). All ELISA results are representative for at least three independent experiments using proteins derived from three independent transfections.</p
Enhancement of α-Helix Mimicry by an α/β-Peptide Foldamer via Incorporation of a Dense Ionic Side-Chain Array
We report a new method for preorganization of α/β-peptide
helices, based on the use of a dense array of acidic and basic side
chains. Previously we have used cyclically constrained β residues
to promote α/β-peptide helicity; here we show that an
engineered ion pair array can be comparably effective, as indicated
by mimicry of the CHR domain of HIV protein gp41. The new design is
effective in biochemical and cell-based infectivity assays; however,
the resulting α/β-peptide is susceptible to proteolysis.
This susceptibility was addressed via introduction of a few cyclic
β residues near the cleavage site, to produce the most stable,
effective α/β-peptide gp41 CHR analogue identified. Crystal
structures of an α- and α/β-peptide (each involved
in a gp41-mimetic helix bundle) that contain the dense acid/base residue
array manifest disorder in the ionic side chains, but there is little
side-chain disorder in analogous α- and α/β-peptide
structures with a sparser ionic side-chain array. These observations
suggest that dense arrays of complementary acidic and basic residues
can provide conformational stabilization via Coulombic attractions
that do not require entropically costly ordering of side chains
SHIV Plasma Viral Loads in MVC-treated and comparator macaques.
<p>SHIV Plasma Viral Loads in MVC-treated and comparator macaques.</p
The diversity of the infecting SHIV-162P3 inoculum.
<p>(A) A maximum likelihood tree constructed with 42 independent full-length clones isolated from the infecting SHIV-162P3 inoculum. An unrooted tree layout is displayed. The horizontal scale bar represents genetic distance. (B) Entropy plot of inoculum diversity as a function of nucleotide position.</p
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