Gjertrud Maurstad

The Breast Cancer-Associated Glycoforms of MUC1, MUC1-Tn and sialyl-Tn, Are Expressed in COSMC Wild-Type Cells and Bind the C-Type Lectin MGL

Aberrant glycosylation occurs in the majority of human cancers and changes in mucin-type O-glycosylation are key events that play a role in the induction of invasion and metastases. These changes generate novel cancer-specific glyco-antigens that can interact with cells of the immune system through carbohydrate binding lectins. Two glyco-epitopes that are found expressed by many carcinomas are Tn (GalNAc-Ser/Thr) and STn (NeuAcα2,6GalNAc-Ser/Thr). These glycans can be carried on many mucin-type glycoproteins including MUC1. We show that the majority of breast cancers carry Tn within the same cell and in close proximity to extended glycan T (Galβ1,3GalNAc) the addition of Gal to the GalNAc being catalysed by the T synthase. The presence of active T synthase suggests that loss of the private chaperone for T synthase, COSMC, does not explain the expression of Tn and STn in breast cancer cells. We show that MUC1 carrying both Tn or STn can bind to the C-type lectin MGL and using atomic force microscopy show that they bind to MGL with a similar deadadhesion force. Tumour associated STn is associated with poor prognosis and resistance to chemotherapy in breast carcinomas, inhibition of DC maturation, DC apoptosis and inhibition of NK activity. As engagement of MGL in the absence of TLR triggering may lead to anergy, the binding of MUC1-STn to MGL may be in part responsible for some of the characteristics of STn expressing tumours.

Dehydration stability of amyloid fibrils studied by AFM

Atomic force microscopy was used to investigate the stability of dehydrated amyloid fibrils formed by human islet polypeptide (IAPP) and Aβ(1–42) peptides. IAPP amyloid fibrils were imaged in liquid (hydrated state) and in air (dehydrated). In addition, fibrils dried on the mica surface were rehydrated and re-examined both in liquid and in air (after consecutive redrying). As reported previously, the initial drying process does not result in any major change in the amyloid appearance and the dimensions of the fibrils are preserved. However, when once-dried samples are rehydrated, fibril stability is lost. The fibrils disintegrate into small particles that are attached to the mica surface. This process is further confirmed by studies of the rehydrated samples after drying, on which the morphology of the fibrils is clearly changed. Similar behavior is observed for Aβ(1–42) amyloid fibrils, which are apparently stable on first drying, but disintegrate on rehydration. The observed change indicates that dehydration is causing a change in the internal structure of the amyloid fibrils. This has important implications for studies of amyloid fibrils by other techniques. Due to the potential influence of hydration and sample history on amyloid structure, preparation and study of amyloid samples with controlled humidity requires more consideration.

PEGylated chitosan complexes DNA while improving polyplex colloidal stability and gene transfection efficiency.

Chitosan is widely explored as a gene delivery vehicle due to its ability to condense DNA, facilitate transport, and subsequent release allowing gene expression, as well as protecting the DNA. Here, we investigate the enhancement of chitosan-DNA dispersion stability while maintaining transfection efficacy by PEGylation of chitosan. Molecular properties of fully deacetylated chitosans and degree of PEGylation were investigated with respect to compaction of DNA, stability and transfection efficacy. Each of the three chitosan samples with varying chain lengths was PEGylated at three different degrees. The chitosans with degree of PEGylation from 0.6 to 1.9% made polyplexes with DNA. PBS induced colloidal aggregation of polyplexes with initial radius of about 100 nm observed for nonPEGylated chitosans was suppressed for 1.9% PEGylated chitosans. The observed increase in transfection efficacy coinciding with increased polyplex colloidal stability suggests that aggregation of gene-delivery packages may reduce the transfection efficacy.

Characterizing DNA condensation by structurally different chitosans of variable gene transfer efficacy.

Chitosan can be used as a nonviral gene delivery vector for which DNA condensation and transfection efficacy strongly depend on structural parameters. In this study, we characterized the condensation of DNA by three molecularly tailored chitosans, including linear, trisaccharide substituted-, and self-branched trisaccharide substituted chitosan oligomers. No significant differences could be detected in the hydrodynamic diameters formed by the various chitosans as analyzed by dynamic light scattering. However, atomic force microscopy revealed that self-branched chitosan formed complexes with a higher ratio of globules to rods, and the heights of both globules and rods were larger than for complexes formed by the other chitosans. Using an amino/phosphate ratio of 10, fluorescence correlation spectroscopy measurements showed that self-branched chitosan exhibited a lower fraction (30%) of bound chitosan than the other chitosans. YOYO-1 was a superior fluorescent DNA-label compared to Cy5 and PicoGreen, since labeling with YOYO-1 had least effect on the size and structure of the complexes.

Isothermal titration calorimetry study of the polyelectrolyte complexation of xanthan and chitosan samples of different degree of polymerization

Mixing oppositely charged polyelectrolytes in aqueous solutions leads to the spontaneous formation of polyelectrolyte complexes. Here, we characterize the interaction between xanthan of two different chain lengths, a tri-glucosamine and a chitosan polymer by isothermal titration calorimetry (ITC). Analysis of the experimental thermodynamic data assuming a single set of identical sites indicated both enthalpic and entropic contributions to the overall interaction in the interaction between xanthan and tri-glucosamine. The relative contribution of entropy compared to enthalpy was found to be largest for the shortest chain length of xanthan. Using a chitosan polymer instead of tri-glucosamine gave rise to two different stages in the interaction process. A model where the first stage of the ITC curve represent an initial polyelectrolyte complexation stage followed by aggregation on further titration of chitosan to the xanthan is suggested. Ultrastructure images by applying atomic force microscopy at some selected extents of titration are consistent with the two-stage interpretation of the thermodynamic data.

Delaying cluster growth of ionotropic induced alginate gelation by oligoguluronate.

Alginates form gels in the presence of various divalent ions, such as Ca(2+) that mediate lateral association of chain segments. Various procedures exist that introduce Ca(2+) to yield alginate hydrogels with overall homogeneous or controlled gradients in the concentration profiles. In the present study, the effect of adding oligomers of α-l-guluronic acid (oligoGs) to gelling solutions of alginate was investigated by determination of the cluster growth stimulated by in situ release of Ca(2+). Three different alginate samples varying in fraction of α-l-guluronic acid and molecular weights were employed. The cluster growth was determined for both pure alginates and alginates with two different concentrations of the oligoGs employing dynamic light scattering. The results show that addition of oligoG slows down the cluster growth, the more efficient for the alginates with higher fraction of α-l-guluronic acid, and the higher molecular weight. The efficiency in delaying and slowing the cluster growth induced by added oligoG were discussed in view of the molecular parameters of the alginates. These results show that oligoG can be added to alginate solutions to control the cluster growth and eventually also transition to the gel state. Quantitative relation between the concentration of added oligoG, type and molecular weight of the alginate, and concentration, can be employed as guidelines in tuning alginate cluster growth with specific properties.