Non-invasive pulsed ultrasound quantification of the resolution of basal cell carcinomas after photodynamic therapy.

The probability of local control of basal cell carcinomas (BCC) treated by photodynamic therapy (PDT) depends strongly on lesion thickness, thicker lesions often requiring two treatments. We examine the utility of 20 MHz pulsed ultrasound (US) for the non-invasive measurement of thickness and rate of regression after PDT treatment. PDT was by topically applied 20% aminolaevulinic acid, followed at 6 h by a standard 100 J/cm(2) of 630 nm light. Patients ( n=60) were selected as being difficult to treat with existing modalities for reasons of likely poor quality of healing or of cosmesis in this very largely elderly population. Ultrasound 'A' scans were made immediately before treatment, and at first and subsequent follow-ups. Parameters measured non-invasively for BCC, adjacent normal skin, and for fibroses after previous conventional therapies, were (a) thickness of skin or lesion, (b) linear density of ultrasound echoes and (c) linear density of high-amplitude echoes. Prior to treatment, median skin thickness (to the dermal/subcutaneous boundary) was 2.6 mm (range 1.2-5.7), fibroses 2.5 mm (1.4-5.6) and BCC 1.5 mm (0.5-4.4). Median linear density of echoes for normal skin, fibroses and BCC plus underlying tissue were 5.6, 5.5 and 4.5, respectively, the BCC values being significantly lower ( p=0.002). The corresponding medians for high-amplitude echoes were 1.9, 1.9 and 1.1 (skin or fibrosis versus BCC, p=0.001). Patients whose BCCs appeared clinically to be controlled at up to 220 days after a single treatment, all had values of ultrasound parameters corresponding to skin/fibrosis and were significantly different from measurements on the same site prior to treatment. Patients whose tumours appeared to be reverting to the original BCC ultrasound pattern were subsequently found to be recurring as judged clinically. Non-invasive pulsed ultrasound indicates that rates of resolution vary widely between BCC of similar initial thickness and that the probability of clearance of BCC by PDT is determined largely by the deepest, sometimes small, regions within a lesion, with the overall area being relatively unimportant.

Combined strong anion-exchange HPLC and PAGE approach for the purification of heparan sulphate oligosaccharides.

Heparan sulphates are highly sulphated linear polysaccharides involved in many cellular functions. Their biological properties stem from their ability to interact with a wide range of proteins. An increasing number of studies, using heparan sulphate-derived oligosaccharides, suggest that specific structural features within the polysaccharide are responsible for ligand recognition and regulation. In the present study, we show that strong anion-exchange HPLC alone, a commonly used technique for purification of heparan sulphate-derived oligosaccharides, may not permit the isolation of highly pure heparan sulphate oligosaccharide species. This was determined by PAGE analysis of hexa-, octa- and decasaccharide samples deemed to be pure by strong anion-exchange HPLC. In addition, subtle differences in the positioning of sulphate groups within heparan sulphate hexasaccharides were impossible to detect by strong anion-exchange HPLC. PAGE analysis on the other hand afforded excellent resolution of these structural isomers. The precise positioning of specific sulphate groups has been implicated in determining the specificity of heparan sulphate interactions and biological activities; hence, the purification of oligosaccharide species that differ in this way becomes an important issue. In this study, we have used strong anion-exchange HPLC and PAGE techniques to allow production of the homogeneous heparan sulphate oligosaccharide species that will be required for the detailed study of structure/activity relationships.

Regulation of FGF-1 mitogenic activity by heparan sulfate oligosaccharides is dependent on specific structural features: differential requirements for the modulation of FGF-1 and FGF-2.

The interaction of heparan sulfate (HS) (and the closely related molecule heparin) with FGF-1 is a requirement for enabling the growth factor to activate its cell surface tyrosine kinase receptor. However, little is known about the regulatory role of naturally occurring cell surface HS in FGF-1 activation. We have addressed this issue by utilizing a library of HS oligosaccharides, which are defined in both length and sulfate content. Mitogenic activation assays using these oligosaccharides showed that HS contained both FGF-1 activatory and inhibitory sugar sequences. Further analysis of these oligosaccharides showed a clear correlation between FGF-1 promoting activity and their 6-O-sulfate content. The results, in particular with the dodecasaccharide sequences, suggested that specific positioning of 6-O-sulfate groups may be required for the promotion of FGF-1 mitogenic activity. This may also be true for 2-O-sulfate groups though the evidence was not as conclusive. Differential activation of FGF-1 and FGF-2 was also observed and found to be mediated by both oligosaccharide length and sulfation pattern, with different specific O-sulfate positioning being implicated for the promotion of different growth factors. These results suggest that variation and tight control of the fine structure of HS may allow cells to not only control their positive/negative responses to individual FGFs but also to change specificity towards promotion of different members of the FGF family.

Monomer complexes of basic fibroblast growth factor and heparan sulfate oligosaccharides are the minimal functional unit for cell activation.

The interaction of basic fibroblast growth factor (bFGF) with heparan sulfate (HS)/heparin has been shown to strongly enhance the activity of the growth factor although the mechanism of activation is unclear. We have addressed the issue of the minimal stoichiometry of an active HS oligosaccharide.bFGF complex by chemically cross-linking the two components to form novel covalent conjugates. The cross-linking procedure produced both monomeric and dimeric bFGF. oligosaccharide complexes, which were purified to homogeneity. Dimer conjugates were shown to have been formed as a result of disulfide bridging of monomer conjugates. These monomer conjugates were subsequently found to be biologically active in a mitogenesis assay. We therefore conclude that a monomeric bFGF.oligosaccharide complex is the minimal functional unit required for mitogenic stimulation.

Sequence analysis of heparan sulphate and heparin oligosaccharides.

The biological activity of heparan sulphate (HS) and heparin largely depends on internal oligosaccharide sequences that provide specific binding sites for an extensive range of proteins. Identification of such structures is crucial for the complete understanding of glycosaminoglycan (GAG)-protein interactions. We describe here a simple method of sequence analysis relying on the specific tagging of the sugar reducing end by 3H radiolabelling, the combination of chemical scission and specific enzymic digestion to generate intermediate fragments, and the analysis of the generated products by strong-anion-exchange HPLC. We present full sequence data on microgram quantities of four unknown oligosaccharides (three HS-derived hexasaccharides and one heparin-derived octasaccharide) which illustrate the utility and relative simplicity of the technique. The results clearly show that it is also possible to read sequences of inhomogeneous preparations. Application of this technique to biologically active oligosaccharides should accelerate progress in the understanding of HS and heparin structure-function relationships and provide new insights into the primary structure of these polysaccharides.

Heparan sulfate oligosaccharides require 6-O-sulfation for promotion of basic fibroblast growth factor mitogenic activity.

The interaction of heparan sulfate (HS) with basic fibroblast growth factor (bFGF) is influential in enabling the growth factor to bind to its cell surface tyrosine kinase receptor. In this study, we have investigated further the structural properties of HS required to mediate the activity of bFGF in a mitogenic assay. We have prepared a library of heparinase III-generated HS oligosaccharides fractionated by both their size (dp6-dp12) and sulfate content. The ability of these oligosaccharides to activate bFGF in a mitogenic assay was then correlated with their length and disaccharide composition. All octa- and hexasaccharide fractions tested were unable to activate bFGF. Dodeca- and decasaccharide fractions were found to contain both activating and non-activating oligosaccharides, and showed a clear correlation between total sulfate content and the level of activatory activity. Disaccharide analysis of a range of dodeca- and decasaccharide fractions showed that both activating and non-activating oligosaccharides were composed mainly of N-sulfated and IdoA(2S)-containing disaccharides. The only significant difference between activating and non-activating oligosaccharides was the content of 6-O-sulfated disaccharides, in particular the disaccharide IdoA(2S)alpha1,4GlcNSO3(6S). These results show that there is a requirement for 6-O-sulfation of N-sulfated glucosamine residues, in addition to the 2-O-sulfation of IdoA, for the promotion of bFGF mitogenic activity by naturally occurring HS oligosaccharides. Analysis of the structure-activity relationships in the dodecasaccharide fractions in particular, suggests that a minimum bFGF activation sequence exists which is dependent on the positioning of at least one 6-O-sulfate group.

Endothelial and fibroblast cell-derived heparan sulphate bind with differing affinity to basic fibroblast growth factor.

Heparan sulphate from endothelial cells (ECHS) has been shown to bind to bFGF with a lower affinity than that seen for 3T3 fibroblast HS (FHS). To investigate the structural reasons for the low affinity binding of ECHS to bFGF, enzymatic degradation of intact ECHS and FHS chains was undertaken. Filter binding assays showed ECHS heparinase III-resistant fragments 6-7 disaccharides in length and had affinity for bFGF equivalent to that of the intact ECHS chains. The largest resistant fragments from FHS, again 6-7 disaccharides in length, bound to bFGF with a similar affinity to the largest ECHS oligosaccharides, and they therefore have considerably lower affinity than seen for the intact FHS chains. Disaccharide compositional analysis of both ECHS and FHS oligosaccharides showed them to contain similar amounts of 2-O-, 6-O-, and N-sulphated disaccharides. These results suggest that the sulphation pattern within sulphated HS domains and their overall length are not the sole contributors to the binding of intact HS chains to bFGF. It is suggested that domain organisation and frequency of occurrence of large heparinase III-resistant oligosaccharides within intact chains play an important role not only in governing the maximum observed binding affinity of intact chains in the assay system used, but also in the regulation of other biological properties of HS.

Molecular attributes of bovine aortic endothelial cell heparan sulfate.

Heparan sulfate (HS) secreted into the medium of bovine aortic endothelial cell (BAEC) cultures was subjected to chemical and enzymatic degradation followed by analysis using gel-filtration and ion-exchange chromatography. Treatment with HNO2 showed that 41% of the disaccharides were N-sulfated. Degradation by Heparin lyases I (Hep I) showed that 8-9% of the disaccharides contained IdoA(2S) residues. Heparin lyase III (Hep III) degradation produced mainly disaccharides with 67% of the molecules glycosidic linkages susceptible to cleavage. Further degradation of Hep III-resistant fragments with Hep I showed that IdoA(2S) residues were predominantly positioned centrally within the repeating GlcNSO3(+/- 6S)alpha 1-4IdoA containing domains. Digestion with a mixture of Heparin lyases I, II and III degraded the molecule almost entirely to disaccharides, with small amounts of tetrasaccharides containing resistant linkages, suggesting the presence of 3-O sulfated GlcNSO3. Further analysis of the disaccharide products by ion-exchange chromatography and comparison with the data from single enzymatic digestion, allowed an estimate of the disaccharide composition to be made. The results suggest an ordered arrangement of structural domains; however, variations in the structure of these domains results in a heterogeneous population of HS chains. It is suggested that biosynthetic differences in HS structure may act as a regulator of bFGF induced cellular responses.

Irradiation of bovine aortic endothelial cells enhances the synthesis and secretion of sulphated glycosaminoglycans.

The effect of X-irradiation on the synthesis of heparan sulphate (HS) and chondroitin/dermatan sulphate (CS/DS) by bovine aortic endothelial cells (BAEC), was studied by measuring the incorporation of [35S]sulphate and [3H]glucosamine into newly synthesized glycosaminoglycan (GAG) chains. Medium extracts from irradiated cultures (5Gy) were found to contain approx. 130% more HS and 200% more CS/DS than unirradiated controls. Smaller increases were observed in cellular extracts, irradiated cultures (5Gy) containing approx. 60% more HS and 100% more CS/DS than unirradiated controls. Structural studies showed no significant changes occurred upon irradiation in either the amounts or distribution of N- and O-sulphate groups in the HS molecule. Values for N-sulphation of 41.1% control and 41.5% irradiated (5Gy) were obtained, the corresponding values for O-sulphation being 19.9% control and 20.2% irradiated. Isotope incorporation data indicated that sulphation of CS/DS may decrease after irradiation, however, analysis of chondroitin ABC lyase derived disaccharides showed no changes in the proportion of non-sulphated and O-sulphated disaccharides. The present study indicates that X-irradiation stimulates the synthesis and secretion of HS and CS/DS proteoglycans (PGs) by BAEC. This could be relevant to many features which are found to be indicative of radiation-induced damage.

Co-operative binding of ribosomal proteins to an erythromycin affinity column.

The binding of the E. coli ribosomal proteins L4, L5 and L21 to an erythromycin affinity column has been found to be co-operative. Binding does not occur in the absence of other ribosomal proteins.