The use of contextual learning to teach biochemistry to dietetic students.

This article describes the use of contextualized and "blended" learning to teach biochemistry to dietetic students during the second year of their professional training in a 4-year undergraduate degree (Bachelor of Nutrition and Dietetics). Contextualized content was used to engage students and motivate them to learn biochemistry, which many perceived as a "hard" subject. Contextualized learning presented via problem solving exercises, case-studies, and by the use of virtual subjects in laboratory class introduced content material through real life situations highly relevant to their later clinical practice. A "blended" curriculum where content was presented in a number of different modalities (on-line, on CD, or face-to-face in small/large groups and in tutorials/lectures/laboratory class) further enhanced contextualized learning by providing a range of learning modalities catering to different student learning styles. The on-line and CD material also allowed student self-assessment of learning progress through interactive quizzes in varied assessment formats, where feedback was often immediate. Student responses to this biochemistry course have been positive with 89% finding it intellectually stimulating.

Partial characterization of matrix components interacting with cartilage proteoglycans.

The charge content of aqueous suspensions of milled cartilage samples was determined by a colloid titration technique using a particle charge detector, and the data were compared with estimates from chemical analyses. Results indicated a close correlation between charge content determined by titration and that estimated by chemical analyses for samples of nasal septa only (a nonarticular cartilage). Such correlation did not hold for articular cartilages (metacarpalphalangeal joint and patella); extraction of these tissues with 0.1 or 1.2 M NaCl markedly increased the availability of the negative groups. Protein analysis, by SDS--PAGE, of the 1.2 M extracts indicated the presence of basic proteins, some of collagenous origin, such as chondrocalcin and proline-arginine-rich protein, and some of noncollagenous proteins such as pleiotrophin and histone-H2b. These data thus suggest electrostatic interactions between these basic proteins and the negative groups of proteoglycans. Such interactions would have an important effect on the osmotic properties and in the organization of cartilage.

Dependence of salt concentration on glycosaminoglycan-lysozyme interactions in cartilage.

The cationic protein, lysozyme, has an extracellular distribution in cartilage but its precise role in this tissue has not yet been established. This study describes the dependence of salt concentration on the binding properties of lysozyme isoforms of different cationic charges, isolated from bovine cartilage, to the two major and structurally similar glycosaminoglycans of cartilage, i.e., chondroitin sulfate and hyaluronan. The binding of most cartilage lysozyme isoforms and hen egg-white lysozyme (control) to chondroitin sulfate and hyaluronan linked to agarose supports displayed optimal levels at approximately 20 and 5-10 mM salt, respectively, but decreased at both lower and higher salt concentrations indicating the electrostatic nature of the interactions. However, optimal binding of the most cationic lysozyme isoform to chondroitin sulfate occurred at 60 mM salt, with significant binding remaining at 150 mM. This isoform also showed binding to hyaluronan up to 60 mM salt, while for the other isoforms binding was observed only up to 150 and 40 mM salt for chondroitin sulfate and hyaluronan, respectively. The low salt concentrations at which these interactions occur are likely to exist in cartilage as shown from equilibrium dialysis studies performed using solutions of chondroitin sulfate (up to 10%, a concentration likely to occur in cartilage). From Scatchard analysis, the affinity of binding of all lysozymes to chondroitin sulfate was similar (Kd = 10(-6) M) and slightly lower than their binding to hyaluronan (Kd = 10(-7) M) of similar molecular mass.

Temporal changes in charge content of cultured chondrocytes from bovine cartilaginous tissues.

The effective charge content of the pericellular matrix of chondrocytes has been determined while the matrix is being synthesized by cells grown in culture for several weeks. The data were compared with estimates determined by chemical analysis. When measurements were performed after digestion of the matrix with papain, there was close agreement between results obtained from both techniques for proteoglycans synthesized by chondrocytes from nasal septum (a non-articular cartilage). By contrast, no such agreement was observed for proteoglycans synthesized by chondrocytes from articular cartilage, even after solubilization of the matrix with papain. While the charge calculated from chemical analysis showed a constant increase with time in culture, that measured by colloid titration showed a cyclical pattern, with maximal values occurring on days 7 and 24 of culture and a minimal value on day 14. This inability to detect all negative groups present in the matrix synthesized by articular chondrocytes would suggest the involvement of these groups in electrostatic interactions. Partial characterization of proteins synthesized by the pericellular matrix indicates that the decrease in charge content observed on day 14 could not be attributed to proteins of a particular molecular mass but possibly to an increase in the total amount of protein present. It is concluded that the marked difference in the availability of negative groups between chondrocytes cultured from articular and non-articular cartilages may reflect differences in the interaction of these negative groups with matrix components; these differences would lead to the distinct structural organization of these two cartilaginous tissues which possess different mechanical functions.

Purification, characterization, and biosynthesis of bovine cartilage lysozyme isoforms.

The cationic protein, lysozyme, has an extracellular distribution in cartilage; however, its biological role in this tissue still remains unclear. This study describes a simple and high yielding procedure for the purification of four novel isoforms of lysozyme from the functionally different articular (metacarpalphalangeal joint) and nonarticular (nasal septum) bovine cartilages. Chromatography of the cartilage extracts on S-Sepharose revealed the presence of four major lysozyme active peaks each of which was further purified to homogeneity by gel filtration and reversed-phase chromatography. Each peak yielded a different molecular mass when analyzed by ion spray mass spectrometry, and material isolated from either cartilage source displayed an identical molecular mass for each lysozyme preparation. N-terminal amino acid sequence and amino acid composition analyses confirmed the presence of four novel lysozyme isoforms in both bovine articular and nonarticular cartilages. The lytic activity of each lysozyme isoform toward Micrococcus lysodeikticus was dependent on both the ionic strength and pH of the buffer, where an increase in activity accompanied an increase in ionic strength. The lysozymes were shown to be synthesized by chondrocytes in vitro, which in addition to the relatively high chemical amounts of lysozyme present in cartilage, would suggest that this small cationic protein has some as yet undetermined biological role within the cartilage extracellular matrix.

Determination of the charge content at the surface of cells using a colloid titration technique.

A colloid titration technique has been used to estimate the surface charge content of three distinct cell types of differing surface charge characteristics, i.e., human red blood cells, the surface of which is studded with sialic acid residues, endothelial cells which are surrounded by a thick glycocalyx, and chondrocytes which, when grown at high cell density for several weeks, synthesize a dense pericellular matrix similar to that observed in cartilaginous tissues. Estimates of the charge content obtained for human erythrocyte ghosts and cultured endothelial cells are in good agreement with the charge determined by chemical analyses. On the other hand, the charge at the surface of chondrocytes represented only a fraction of that calculated from measurements of the glycosaminoglycan content (15% by Day 12 in culture); close correlation between charge and chemical amount could be obtained only after digestion of the cell layer with collagenase and papain indicating the possible electrostatic involvement of the negative groups of the glycosaminoglycan chains with basic proteins. Thus, the colloid titration technique may provide a simple and sensitive tool to study the interactions occurring between the extracellular matrix components while the matrix is being formed and to establish the chemical nature of the molecules contributing to the cell surface charge.

Binding properties of glycosaminoglycans to lysozyme--effect of salt and molecular weight.

The cationic protein, lysozyme, is present in cartilage but its precise role in this tissue has not yet been established. This study shows that two major and structurally similar glycosaminoglycans (GAGs) of cartilage, i.e., chondroitin sulfate (CS) and hyaluronan (HA) interact with lysozyme at salt concentrations up to 40 mM. Such a low salt concentration is likely to occur in cartilage due to exclusion of microions by the high charge density of the proteoglycans (PGs). The affinity of binding to lysozyme increases with increasing molecular weight of HA and is higher for HA (Kd = 1-2 x 10(-8) M and 0.5-1 x 10(-7) M for HA of relative molecular mass of 4 x 10(5) and 5 x 10(4), respectively) than for CS (Kd = 1 x 10(-6) M). The binding displays optimal levels at around 20 mM but decreases at both lower and higher salt concentrations. This dependence of binding on salt concentration resembles that of the enzymic activity of lysozyme for its natural substrate, murein, which is structurally similar to HA/CS. The increase in binding up to 20 mM salt is characteristic for HA/CS-lysozyme interaction as such an effect was not observed in the interaction of heparin with lysozyme or of GAGs with serum albumin. Binding of HA to lysozyme was inhibited by various polyanions but not by uncharged macromolecules, indicating the electrostatic nature of the interaction. The dependence of binding on salt concentration obtained in systems where lysozyme is linked to an agarose support and the GAG is free in solution is similar to that determined when both macromolecules are free in solution; however, the number of GAG disaccharides bound per mole lysozyme increases significantly in the latter system, indicating a marked artifactual steric hindrance effect in the former.

The measurement of negative charge content in cartilage using a colloid titration technique.

A colloid titration technique has been used to determine the sulfate and carboxylate content of various glycosaminoglycans and has been validated by comparing the results with data obtained using well-established techniques. The method has been applied to the measurement of the negative charge content of cartilage slices at various depths from the articular surface and to the determination of sulfate and carboxylate contents in bovine nasal septa. Titrations of nasal septa were performed on milled cartilage, on cartilage digested with papain and on proteoglycans purified by cesium chloride gradient centrifugation of guanidinium chloride extracts. The sulfate content was similar for all three preparations (0.5 mu eq per milligram dry cartilage). However, the carboxylate content determined on milled cartilage was 40% higher than that obtained for cartilage digested with papain or for purified proteoglycans; this implies the possible contribution of carboxyl groups from structural glycoproteins present in the extracellular matrix. The carboxylate content determined on purified proteoglycans was in excellent agreement with values calculated from chemical analyses.

Binding of hyaluronan to lysozyme at various pHs and salt concentrations.

Binding of hyaluronan (HA) to lysozyme immobilized on Sepharose-6B was investigated as a function of pH and NaCl concentration. High affinity binding (Kd = 1.0-2.0 x 10(-8) M) was observed at pH 7.5 and at 10-50 mM NaCl; the number of moles of HA bound to lysozyme was twice as high at 30 mM NaCl as at 10 mM. No specific binding was observed at and above 100 mM NaCl. Binding was suppressed in the presence of chaotropic agents such as guanidinium chloride and urea. These results suggest that binding between HA and lysozyme can occur in the extracellular matrix where an electrolyte concentration as low as 50 mM could be expected due to ionic exclusion by the highly negative charge concentration arising from the polyanions present.

Evaluation of nonideality from gel chromatographic partition coefficients. A technique with greater versatility than equilibrium dialysis.

Frontal gel chromatography has been used to measure partition coefficients which enable a quantitative evaluation of the thermodynamic nonideality of small solutes generated by the presence of high concentrations of macromolecular solutes. Equivalence of results obtained by the present method and by equilibrium dialysis is demonstrated in a comparison of results for dextran sulfate-NaCl and dextran-sorbitol systems. Interaction coefficients obtained for dextran-sorbitol and protein-polyethylene glycol 4000 systems yields results which are in reasonable agreement with those predicted on the statistical-mechanical basis of excluded volume. Because of its greater versatility in regard to the range of systems that may be studied, the frontal gel chromatographic procedure is likely to be of particular value for the quantitative characterization of thermodynamic nonideality arising from excluded volume effects in concentrated mixtures of macromolecular solutes.

A sensitive and specific in vitro bioassay method for the measurement of follicle-stimulating hormone activity.

An in vitro bioassay method for hFSH is presented. The method is based on the principles previously described by Dorrington et al. (1976b) and involves the assay of oestradiol produced from 19-hydroxyandrostenedione by dispersed Sertoli cells of 10-day old rats when cultured in the presence of graded doses of FSH. Using the 1st International Reference Preparation for human pituitary gonadotrophins (FSH and LH/ICSH) for bioassay (code no. 69/104) as standard, the useful range of the method is from 0.5 to 32 mIU/chamber (2 to 128 mIU/ml). The sensitivity of the method is 0.5 mIU/chamger. The mean index of precision (lambda) obtained from 16 multiple assays over 2 or 3 dose levels was 0.084. Parallelism was obtained between the 69/104 preparation and all preparations under study. The practicability of the proposed assay method is such that 15 preparations at 3 dose levels can be assayed by one person in 3 days. The specificity of the assay was investigated by determining the FSH activity in the following preparations: hFSH alpha- and beta-subunits, hLH, hCG, hTSH, ACTH, human growth hormone (hGH) human prolactin (hPRL) and luteinizing hormone-releasing hormone (LH-RH). The ACTH, hGH, hPRL and LH-RH preparations studied showed no detectable FSH activity in the assay. In the remaining preparations very low levels of FSH activity were found, corresponding to 0.004 to 0.6% of the weight of these preparations when compared with a highly purified hFSH preparation, suggesting that the method is specific for FSH. The possible synergistic or antagonistic influence of the above preparations when assayed in the presence of the 69/104 preparation was also assessed. No evidence of a synergistic or antogonistic effect was found. The assay of the hFSH potencies of a limited number of hFSH preparations of varying purity by the proposed in vitro bioassay, an hFSH radioreceptor method and an hFSH specific radioimmunoassay technique revealed that - although the relationship of the various potencies obtained with each method showed a close agreement - the bioassays yielded the highest potency estimates, and the radioimmunoassays the lowest ones. Since the proposed bioassay method is sensitive and considered to be specific for hFSH activity, it provides a suitable basis for the assessment of the specificity of other in vitro methods (radioreceptor and radioimmunoassay) currently used for detecting low levels of FSH activity.

Biological and immunological characterization of human luteinizing hormone: I. Biological profile in pituitary and plasma samples after electrofocusing.

Pituitary and plasma pools from postmenopausal women and plasma pools from women at midcycle were fractionated by electrofocusing in sucrose density gradients. The biological LH activity was determined in each of the electrofocusing fractions by the use of an in vitro bioassay method. A heterogeneous profile of LH activity was found in both pituitary and plasma samples with a large proportion present within the pH range 6.5-10. In a total of 11 electrofocusing runs 7 main regions of high LH activity were found within this range with mean pI values (+/- SD) of 6.75 +/- 0.08 (n = 6), 7.33 +/- 0.08 (11), 7.80 +/- 0.09 (11), 8.23 +/- 0.10 (11), 8.81 +/- 0.04 (7), 9.17 +/- 0.05 (6) and 9.55 (2). A significantly higher proportion of LH activity was found in the midcycle plasma samples (36%) in the pH regions with mean pI values of 8.81, 9.17 and 9.55 than in postmenopausal plasma (7%) and pituitary extracts (5%). This indicates that the profile of biologically active LH in women in the fertile age is different from that present in postmenopausal women. By detailed fractionation based on narrow pH range studies and the refocusing of specific peak fractions it was shown that each of the regions studied consisted of several peaks of LH activity indicating the presence of a large number of molecular species exhibiting varying degrees of LH activity. The relative proportions of these species showed considerable differences between sources but also between samples from the same source.