230 



M. K. KEECH AND R. REED 



may slide over each other to allow for the alteration 

 in the contour of the animal. No banded structure 

 has been observed, but the fibrils are surrounded by 

 an amorphous material which tends to make precise 

 observation difficult. This latter material is formed 

 into a fibrous honeycomb which surrounds each 

 fibril (fig. 3). It is possible that this material may re- 

 present a mucopolysaccharide; hence it may form 

 an underlying lattice system which may be connected 

 with the mechanism of determining the orientation 

 of the fibrils. 



Region 4 lies just beneath the outermost layer; 

 it sometimes contains a few fibrils but more often 

 consists only of a thin amorphous zone. The outer- 

 most layer, region 5, is composed of a double mem- 

 brane, from which project numerous microvilli 

 about 1500 A long and 500 A in diameter. Reed 

 and Rudall (4) described this region as a corpuscular 

 layer, but observations on transverse sections of the 

 cuticle show clearly that the exterior of the worm is 

 covered by a system of microvilli. This finding may 

 account for the presence of the numerous fine cyto- 

 plasmic processes which penetrate the cuticle, for it 

 is unlikely that the microvilli could exist in an extra- 

 cellular position. 



Previous work has demonstrated that in avian 

 tendon the collagen fibrils of developing tissue in- 

 crease in diameter v^hile there is a relative decrease 



in the amount of interfibrillar material which sur- 

 rounds the fibrils; it has been concluded that this 

 material contains collagen molecules which are depo- 

 sited onto the growing fibrils in the form and packing 

 appropriate to the characteristic fibre diagram of 

 the collagen protein (2). In the present work, it is 

 apparent that the layers of fibrils adjacent to the 

 epidermal cells are of smaller diameter than the 

 main fibrils of the cuticle. If it is assumed that the 

 epidermal cells secrete the precursors of the cuticle, 

 it follows that the internal layers may be composed 

 of fibrils that are in the process of growing. Thus, it 

 may be suggested that, as each fibril layer is formed, 

 the next adjacent layer may determine the orienta- 

 tion of this new layer. On the other hand, it is 

 possible that the precisely arranged cytoplasmic 

 processes may take part in the orientation of the 

 fibril layers. It is hoped that future studies on the 

 regeneration of the cuticle will clarify the method of 

 fibril elaboration and layer formation. 



References 



1. AsTBURY, W. T., Essays on Growth and Form. P. 309 



(1945). 



2. Jackson, S. Fitton, Pioc. Roy. Soc. B. 144, 553 (1956). 



3. Marks, M. H., Bear, R. S., and Blake, C. H., /. E.xptl. 



Zool. Ill, 55 (1949). 



4. Reed, R. and Rudall, K. M., Biochini. Biophys. Acta 2 



7 (1949). 



Further Observations on the Transformation 

 of Collagen Fibrils into ''Elastin'' 



M. K. Keech and R. Reed 



Departments of Medicine and Leather Industries, Leeds University, Leeds 



Xhe dermis consists mainly of collagen (70 80 g 

 per 100 g dry tissue) and a small quantity of elastin, 

 all embedded in the jelly-like ground substance. 

 Previous work (7) on the action of collagenase on 

 human abdominal skin from different age-groups, 

 described a breakdown product called "moth-eaten" 

 fibres (MEF). They figured prominently in colla- 

 genase-treated material derived from persons be- 

 tween the ages of 1 and 20 years, but their numbers 

 steadily decreased as the age of the collagen source 

 increased. At first sight it was difficult to understand 

 the appearance of these large and very dense struc- 

 tures during an enzyme degradation process, so 

 further investigations were undertaken in an effort 

 to establish their identity (8). Identical experiments 

 designed to obtain more information about the 

 "alkali-produced elastin" (APE) described by Bur- 

 ton et al. (I) were also reported. 



Prepared collagen. — Samples of the same substrates used 

 in previous work (5, 6, 7) were employed, i.e., human 

 abdominal skin collagen from persons of different age- 



groups, purified by the method of Neuman (9, 10) as 

 shortened by Keech (5, 6). This material contained a 

 very small quantity of the three morphological varieties 

 of fully-formed elastin described below. 



Preparations of "moth-eaten' fibres (MEF). — Prepared 

 collagen was incubated with collagenase in phosphate 

 buffer (pH 7.4) for 24 hours at 37~C. The remaining 

 material was centrifuged, and the pellet treated one of 

 the following ways: — 



(a) Re-suspended in sterile distilled water, pH 5.6, and 

 heated at 55 C for 1 hour, at 75'C for a further hour and 

 finally at 100 C for 1 hour. 



(h) Re-suspended in a mixture of 1 "o sodium meta- 

 periodate and 1 % NaCI in phthalate buffer at pH 5.0 

 and incubated for I h hours at 37''C. 



((■) Re-suspended in borate buffer (pH 8.8) containing 

 0.2 mg elastase and incubated for 6 hours at 37C. 



(d) Re-suspended in 2 % acetic acid, allowed to stand 

 at room temperature for an hour and then heated at 

 100 C for 1 hour. 



Preparations of alkali-produced ''elastin" (APE). — The 

 method used in previous work (1, 3) to effect the apparent 

 transformation of collagen into elastin was employed, 

 i.e., prepared collagen was incubated in borate buffer 

 (pH 8.8) for 24 hours at 3TC. The centrifuged pellet was 

 then treated as in (a) and (d) above. In addition it was 



