232 



M. K. KEECH AND R. REED 



•.^J^ 



Fig. 3. "Moth-eaten" fibre disintegrating into an "elastin 

 network" (MEFC). Obtained after heating the MEF prepara- 

 tion from a 9-year-old child in water for 1 hour at 55°C and 

 a further hour at 75''C. Magnification 11,000. 



Fig. 4. Another example of a "moth-eaten" fibre conversion 

 structure (MEFC) from the same preparation as fig. 3. 

 Magnification 1 1 ,000. 



The manufactured networks (MN) were found 

 only in the presence of MEFC and probably repre- 

 sent the complete conversion or separated portions 

 of these structures. Similar MN occurred after 

 heating "alkali-produced elastin". 



Collagen was seen only in the adult specimens 

 and exhibited slight "standard" collagenase change. 



3. Effect of other reagents on MEF. A dramatic 

 change from 90 % MEF to 85-90 % large "elastic 

 networks" was produced by 1 % sodium periodate 

 (in buffer pH 5.0) and by elastase (acting at its op- 

 timum pH of 8.8). Also numerous large and small 

 dense bits were noted, either converting into "elas- 

 tin" individually or situated in groups linked together 

 by "elastin". A more prolonged incubation with 

 periodate (3 hrs. at 37°C) caused disappearance of 

 the MEF and elastic structures, but numerous small, 

 round, dense balls were found, similar to those 

 described by Hall et al. (4) as a product of the action 

 of elastase on elastin. Incubation of the starting 

 material in borate buffer pH 8.8 alone did not pro- 

 duce any change, but prolongation of incubation 

 with elastase caused rapid digestion of the "elastic 

 networks". 



Two per cent acetic acid at room temperature 

 produced no macroscopic change but under the 

 electron microscope the dense bits were broken up 

 into smaller pieces and MEFC and MN were seen. 

 After 1 hour at lOO'C the dense bits were markedly 

 decreased, accompanied by a great increase in 

 MEFC, MN and fenestrated sheets. The latter were 

 only seen after treatment with acetic acid, and 

 probably represent MN with a thicker layer of 

 amorphous material than usual. 



4. Effect of various agents on ''alkali-produced 

 elastin"" {APE). Collagen and elastin have always 



been considered as two separate and distinct entities 

 with different chemical and tinctorial properties. 

 But the problem of material apparently intermediate 

 between them, found in senile and pathological 

 skin, has been a repeated source of debate by his- 

 tologists over the past 90 years. Recent work (1,3) 

 has produced biochemical, histological, and electron 

 microscopic evidence that collagen can be converted 

 into "elastin" /// vitro by agents such as periodate and 

 alkaline buffer (pH 8.8). For this reason, the elastase 

 in the present study was incubated at neutral pH in 

 order to avoid the continued production of APE 

 at the optimum pH value of the enzyme 8.8. The 

 "alkali-produced elastin" (APE) proved sensitive to 

 the elastase. 



The addition of 2 % acetic acid to APE led to 

 immediate dispersal of the collagen suspensions 

 which then appeared gelatinous. This contrasted 

 with the lack of macroscopic effect when acetic acid 

 was added to the (collagen-free) suspensions of 

 MEF. Under the electron microscope the collagen 

 exhibited typical acid disintegration and there was 

 a marked reduction of amorphous material and 

 dense bits together with the appearance of MEFC, 

 MN and fenestrated sheets. 



The effect of heat on APE produced a dramatic 

 change to numerous MEFC and MN with total 

 disappearance of the collagen. 



Concurrent biochemical and histological studies 

 on the same material used in this investigation lead 

 us to believe that "moth-eaten" fibres are interme- 

 diate structures, midway between collagen and elas- 

 tin. 



In the rheumatic group of diseases, the fibrous 

 components of the connective tissue exhibit histo- 

 logical damage. Until the reactivity of the various 

 connective tissue components to a wide range of 

 stimuli (physical, chemical or enzymatic) is eluci- 

 dated and their normal interrelationships established, 

 one cannot interpret the pathological tissue found in 

 disease. 



References 



1. Burton, D., Hail, D. A., Keech, M. K., Reed, R., 



Saxl, H., Tlinbriejce, R. E., and Wood, M. J., 

 Nature 176, 966 (1955). 



2. Hall, D. A. and Gardiner, J. E., Biochem, J. 59, 465 



(1955). 



3. Hall, D. A., Keech, M. K., Reed, R., Saxl, H., Tun- 



bridge, R. E., and Wood, M. J., /. Gerontol. 10. 388 

 (1955). 



4. Hall, D. A., Reed, R., and Tunbridge, R. E., E.xptl. 



Cell Research 8, 35 (1955). 



5. Keech, M. K., Anat. Rec. 119, 139 (1954). 



6. — Yale J. Biol, and Med. 26, 295 (1954). 



7. — Ann. Riwiimatic Diseases 14, 19 (1955). 



8. Keech, M. K., Reed, R., and Wood, M. J., /. Pathol. 



Bacterial. 71, 477 (1956). 



9. Neuman, R. E., Ph.D. Thesis, University of Cincinnati. 



(1949). 

 10. — Arch. Biochem. 24, 289 (1949). 



