Specific sizes of hyaluronan oligosaccharides stimulate fibroblast migration and excisional wound repair.

The extracellular matrix polysaccharide hyaluronan (HA) plays a key role in both fibrotic and regenerative tissue repair. Accumulation of high molecular weight HA is typical of regenerative repair, which is associated with minimal inflammation and fibrosis, while fragmentation of HA is typical of postnatal wounds, which heal in the presence of inflammation and transient fibrosis. It is generally considered that HA oligosaccharides and fragments of a wide size range support these processes of adult, fibrotic wound repair yet the consequences of sized HA fragments/oligosaccharides to each repair stage is not well characterized. Here, we compared the effects of native HA, HA oligosaccharide mixtures and individual sizes (4-10 mer oligosaccharides, 5 and, 40 kDa) of HA oligosaccharides and fragments, on fibroblast migration in scratch wound assays and on excisional skin wound repair in vivo. We confirm that 4-10 mer mixtures significantly stimulated scratch wound repair and further report that only the 6 and 8 mer oligosaccharides in this mixture are responsible for this effect. The HA 6 mer promoted wound closure, accumulation of wound M1 and M2 macrophages and the M2 cytokine TGFβ1, but did not increase myofibroblast differentiation. The effect of 6 mer HA on wound closure required both RHAMM and CD44 expression. In contrast, The 40 kDa HA fragment inhibited wound closure, increased the number of wound macrophages but had no effect on TGFβ1 accumulation or subsequent fibrosis. These results show that specific sizes of HA polymer have unique effects on postnatal wound repair. The ability of 6 mer HA to promote wound closure and inflammation resolution without increased myofibroblast differentiation suggests that this HA oligosaccharide could be useful for treatment of delayed or inefficient wound repair where minimal fibrosis is advantageous

Stimulation of the Maltose Transporter ATPase by Unliganded Maltose Binding Protein

ATP hydrolysis by the maltose transporter (MalFGK(2)) is regulated by maltose binding protein (MBP). Binding of maltose to MBP brings about a conformational change from open to closed that leads to a strong stimulation of the MalFGK(2) ATPase. In this study, we address the long-standing but enigmatic observation that unliganded MBP is also able to stimulate MalFGK(2). Although the mechanism of this stimulation is not understood, it is sometimes attributed to a small amount of closed (but unliganded) MBP that may exist in solution. To gain insight into how MBP regulates the MalFGK(2) ATPase, we have investigated whether the open or the closed conformation of MBP is responsible for MalFGK(2) stimulation in the absence of maltose. The effect of MBP concentration on the stimulation of MalFGK(2) was assessed: for unliganded MBP, the apparent K(M) for stimulation of MalFGK(2) was below 1 microM, while for maltose-bound MBP, the K(M) was approximately 15 microM. We show that engineered MBP molecules in which the open-closed equilibrium has been shifted toward the closed conformation have a decreased ability to stimulate MalFGK(2). These results indicate that stimulation of the MalFGK(2) ATPase by unliganded MBP does not proceed through a closed conformation and instead must operate through a different mechanism than stimulation by liganded MBP. One possible explanation is that the open conformation is able to activate the MalFGK(2) ATPase directly

RHAMM and CD44 are both required for stimulation of wound repair by 6mer HA.

<p>Wild type (wt), CD44−/− and RHAMM−/− (Rh−/−) were wounded as described in Material and Methods. Wounds were treated with Collagen I +/−6mer HA as described in Material and Methods. Wound edges were traced and remaining wound areas quantified by image analysis. Graph shows remaining wound area relative to original wound size on day 5 after wounding. Mean±SE of N = 6 wounds.</p

6mer HA fragments accelerate repair of full thickness excisional wounds.

<p>Full thickness excisional wounds were treated with a mixture of Collagen I and either 6mer, 8mer or 10mer 40(1–50 µg/ml) or PBS as described in Material and Methods. A: Wound edges were traced and remaining wound area quantified by image analysis. Graph shows remaining wound area relative to original wound size on day 7 after wounding. Mean±SE of N = 6 wounds. B: Wound closure is completed 2 weeks after wounding. Representative images of 2 weeks old wounds that were stained with Masson’s Trichrome are shown.</p

6mer HA and 40 kDa HA has no significant effect on Smooth muscle actin or Tenascin C expression during wound repair.

<p>A: Cross sections of 7 days old wounds were stained with Smooth muscle actin specific antibodies as described in Materials and Methods. Positive stained area was quantified by image analysis using ImageJ. Graph shows Mean±SE of N = 18 (6 sections, three areas/section). B: Cross sections of 7 days old wounds were stained with Tenascin C specific antibodies as described in Materials and Methods. Positive stained area was quantified by image analysis using ImageJ. Graph shows Mean±SE of N = 18 (6 sections, three areas/section).</p

6mer and 40 kDa HA moderately increase macrophage infiltration during wound repair.

<p>A: Cross sections of 7 days old wounds were stained with INOS specific antibodies as described in Materials and Methods. Positive cells were counted/area granulation tissue. Graph shows Mean±SE of N = 18 (6 sections, three areas/section). B: Cross sections of 7 days old wounds were stained with ARG1 specific antibodies as described in Materials and Methods. Positive cells were counted/area granulation tissue. Graph shows Mean±SE of N = 18 (6 sections, three areas/section).</p

6mer HA significantly increases TGFβ1 accumulation but has no effect on collagen accumulation during wound repair.

<p>A, B: Cross sections of 7 days old wounds were stained with TGFβ1 specific antibodies as described in Materials and Methods. Positive stained area was quantified by image analysis using ImageJ. Graph shows Mean±SE of N = 18 (6 sections, three areas/section). C: Cross sections of 7 days old wounds were stained with Masson’s Trichrome as described in Materials and Methods. Blue staining (Collagen) was quantified by image analysis using ImageJ. Graph shows Mean±SE of N = 18 (6 sections, three areas/section).</p

Application of <i>Aspergillus niger</i> Fumonisin Amine Oxidase (AnFAO) to Detoxify Fumonisin-Contaminated Maize

Fumonisin mycotoxins are a family of secondary metabolites produced by Fusarium verticillioides and related species, as well as some strains of Aspergillus niger. Fumonisin contamination of maize is a concern when grown under hot, dry conditions. When present above regulatory levels, there can be effects on animal health. New tools to reduce the toxicity of maize and maize products with high concentrations of fumonisin are needed. Recently, we reported an amine oxidase (AnFAO) from a fumonisin-producing Aspergillus niger strain capable of oxidatively deaminating intact fumonisins. In this study, AnFAO was used to reduce intact fumonisin concentrations in milled maize flour, whole kernel maize inoculated with fumonisin-producing Fusarium verticillioides, and dried distillers’ grains with solubles (DDGS). The data showed that milled maize flour incubated with 1 µM AnFAO for 1 h resulted in complete deamination of FB1 and FB2. A greater than 90% reduction in FB1–3 concentrations was observed following a simple washing procedure of whole kernel maize in the presence of 1 µM AnFAO for 1 h. Similarly, a ≥86% reduction in FB1–3 concentrations was observed in DDGS after 4 h incubation with 1 µM AnFAO. Finally, we engineered the methylotrophic yeast Pichia pastoris to produce functional AnFAO in both a secreted and intracellular form. These results support the further development and application of AnFAO as a promising tool to remediate fumonisin-contaminated maize and maize products

How does a protein with dual mitotic spindle and extracellular matrix receptor functions affect tumor susceptibility and progression?

The mechanisms responsible for the oncogenic effects of the hyaluronan (HA) receptor and mitotic spindle binding protein, RHAMM, are poorly understood. On one hand, extracellular RHAMM interacts with HA and cellsurface receptors such as CD44 to coordinately activate the MAPK/ERK1,2 pathway, thus contributing to the spread and proliferation of tumor cells. On the other hand, intracellular RHAMM decorates mitotic spindles and is necessary for spindle formation and progression through G2/M and overexpression or loss of RHAMM can result in multipole spindles and chromosome missegregation. The deregulation of these intracellular functions could lead to genomic instability and fuel tumor progression. This suggests that both extracellular and intracellular RHAMM can promote tumor progression. Intracellular RHAMM can bind directly to ERK1 to form complexes with ERK2, MEK1 and ERK1,2 substrates, and we present a model whereby RHAMM's function is as a scaffold protein, controlling activation and targeting of ERK1,2 to specific substrates