Hyaluronidases synthesis

As an extension of the HA film approach, Yun and coworkers [32] synthesized hyaluronan microspheres using the chemistry described above, but the synthesis was completed in emulsion in one step, yielding 5- to 20-pm microspheres. These microspheres were found to be biodegradable and released three times more pDNA when incubated with hyaluronidase in PBS (phosphate buffered saline) solution (vs enzyme-free PBS). As in the case of films, DNA release from the microspheres was dependent on the DNA loading. DNA-HA microspheres were not directly used for transfection instead, DNA obtained from release experiments was used in transfection of Chinese hamster ovary (CHO) cells using Lipofectamine. The relative levels of transfection over time had the same trend as DNA release from the DNA-HA microspheres and confirmed that released DNA is bioactive. 

Cultures of isolated keratinocytes have facilitated the study of epithelial HA metabolism. Basal keratinocytes synthesize copious quantities of HA. When Ca++ of the culture medium is increased, from 0.05 to 1.20 mM, these cells begin to differentiate, HA synthesis levels drop,148 and there is an onset of hyaluronidase activity.149 This increase in calcium that appears to simulate in culture the natural in situ differentiation of basal keratinocytes parallels the increasing calcium gradient observed in the epidermis. There may be intracellular stores of calcium that are released as keratinocytes mature. 

Larnier, C. et al., Effect of testicular hyaluronidase on hyaluronate synthesis by human skin fibroblasts in culture, Biochim. Biophys. Acta, 1014, 145, 1989. 

Kobayashi S, Fujikawa S, Ohmae M. Enzymatic synthesis of chondroitin and its derivatives catalyzed by hyaluronidase. J. Am. Chem. Soc. 2003 125 14357-14369. 

At the cellular level, bursts of HA synthesis correlate with the onset of mitosis [34,35,63]. This disengages the cell from the ECM and tissue organization, and prepares the cell for the semi-autonomous situation required for cell division. At the completion of mitosis, or at the beginning of Go phase of the cell cycle, a burst of hyaluronidase expression may occur, removing the shell of pericellular HA, preparing the cell for re-association with the ECM... 

Preliminary evidence for the existence of such an organelle comes from a variety of sources. Treatment of cells with low concentrations of hyaluronidase stimulates anomalous levels of HA synthesis, suggesting that some feedback mechanisms exist that instruct the cells in the status of HA metabolism [102,103]. Treating cells with even higher concentrations of hyaluronidase stimulates levels of expression of the predominant HA receptor, CD44 [104,105]. 

Furthermore Kobayashi et al. were able to show that hyaluronidase is able to catalyze the in vitro synthesis of hyaluronan [228], chondroitin [229], chondroitin sulfate [230], their derivatives [229, 231] and of non-natural GAGs [232]. 

There is strong evidence to indicate that ascorbic acid is involved in some way in the synthesis of physiological hyaluronidase inhibitor. A strong suggestion to this effect is provided by the manifestations of scurvy, resulting... 

The present paper focuses on precision synthesis of natural HA (46) and Ch (47) with well-defined structures of biological importance, via hyaluronidase-catalyzed polymerization of sugar oxazoline derivatives (Scheme 1). Similarly, unnatural Chs (6) were prepared using the same enzyme (47) (Scheme 2). These reactions provide a facile and efficient approach to synthesis of GAGs with well-defined structures. 

Figure 2.6 Synthesis ofhyaluronan by wounded HPMCs [56], (A) HA accumulation at the times indicated was determined according to the amount of label incorporated into papain-digested and hyaluronidase-resistant pH] macromolecules (B)The control and injured cultures were pulse labelled with pH]-glucosamine for the time indicated. The rate of HA synthesis is expressed as PHj-HA dpm/h. Injured cells (m) are compared with control experiments (n). Reprinted by permission from Macmillan Publishers Ltd Kidney International from ref [56], copyright (2000)...

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