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Sulfhydryl and disulfide groups in the cuticle of Ascaris lumbricoides var. suis
EXPERIMENTAL
PARASITOLOGY
Sulfhydryl
10,
263-267 (1960)
and Disulfide Groups in the Cuticle of Ascaris lurnbricoides var. suis Luis
M. Carbonell
Seccidn Medicina,
Instituto Apartado
(Submitted
and
Rafael
Apitz
Venezolano de Investigaciones 1827, Caracas, Venezuela for publication,
26 April
C. Cienti$cas,
1960)
The reactions obtained for sulfhydryl and disulfide groups in the cuticle of A.scaris Zumbricoides have demonstrated the transformation of sulfhydryls into disulfides. The -SH groups appear in the anhistous region to form a type of thin fibrils, more dense towards the periphery. The -SS and -SH groups coincide in the squamous region.
Hyman (1951), in her studies of nematodes, disclosed the presence of several regions in the cuticle of Ascaris lumbricoides: i.e. a cortex, a matrix, a fibrous and a basal layer. A study of the distribution of the sulfhydryl and disulfide groups is herein reported. No clear correlation between morphology and said distribution was found. A new histochemical description of the cuticle of Ascaris is made on the basis of the aforementioned groups. MATERIAL
AND METHODS
Specimens of A. lumbricoides var. suis, male and female, were obtained alive from the slaughterhouse, taken to the laboratory in 1% NaCl, dissected, fixed in 1% trichloroacetic acid in 80 % ethyl alcohol and embedded in vacua in paraffin. Longitudinal and transverse sections were cut at 5 p. The following reactions were used to demonstrate the -SH groups : ChevremontFrederic, RSR-SH (red sulfhydryl reagent) (Pearse, 1954) and DDD-SH (2,2’- Dihydroxy-6,6’-Dinaphtil-Disulfide) (Barrnett and Seligman, 1952). DDD-SH, inhibited with 18.6 % iodo-acetic acid at pH 8 and reactivated with 10 % potassium cyanide (Gurr, 1958) was used for the -SS group, as well as hematoxylin-eosin, Gomori’s trichrome (Gomori, 1950), phase contrast and birefringence.
RESULTS
In longitudinal section a well contrasted region, 1.6 p in thickness, is seen at the periphery under oil immersion phase contrast (Fig. 1). This region for which we have devised the name of “squamous region,” is formed by felt-like, angular-ended structures, corrugated for the most part and separated from one another by small noncontrasted interstices. The part immediately below, which we call “sub-squamous region,” is 10.6 /I in thickness and exhibits a negative contrast and a fringed appearance along its From this tract emerge internal tract. slightly contrasted filaments encroaching upon the outermost portion of the “anhistous region” which is 33-p thick. No contrast or structures are seen in this region. The fourth region, called the “fibrous region” can be divided into three layers: i.e. outer, central, and inner layer. The outer layer shows fibres in transverse section with a regular arrangement; the central layer contains zones which are contrasted in relation to the other layers described above; the inner layer shows a low contrast. The transverse sections (Fig. 2) examined under phase contrast demonstrate a continuous, highly contrasted squamous region. The sub-squamous region shows no fringes and has a spongy appearance. The anhistous region, unlike the fibrous one which has
263
264
CARBONELL
FIG. 1. This figure and all that follow are photomicrographs of the -SH and -SS groups in the cuticle of Ascaris lumbricoides var. suis. Phase contrast. Longitudinal section. 400X. A: Squamous region. B: Sub-squamous region. C: Anhistous region. D: Fibrous region.
AND
APITZ
C.
FIG. 2. Phase contrast. Transverse section. 400X. A: Squamous region. B: Sub-squamous region. C : Anhistous region. D : Fibrous region.
the periphery of the squamous region. This reaction in this region shows two positive layers, the positivity decreasing inwards till TABLE I it becomes completely negative, further reDistribution of Sulfhydryl and Disul$de Groups vealing a homogeneous, entirely negative in the Cuticle of Ascaris lumbricoides var. suis region. The sub-squamous region is perfectly recognizable but, like the filaments, is less co positive than the squamous region. The an)Y; .8 82 histous region is completely negative. PosiF4 tive portions are seen in the fibrous region, at regular intervals, all along the three laySquamous region ++ f ers. The inner layer shows a positive reaction Sub-squamous region f + ++ Anhistous region bhroughout, but not so positive as in the + Fibrous region squamous region. outer layer + ++ DDD-SH in transverse section (Fig. 4) central layer + ++ CL demonstrates a strongly positive reaction in inner layer ++ (1) the squamous region. The sub-squamous region is slightly positive and not too visible. (1) Positive in regular-banded structure throughout the layer. The anhistous region is negative, while the ++ Strongly positive. fibrous region is positive throughout but + Positive. weaker in the central layer. - Negative. Chevremont-Frederic -SH in transverse sections shows a distributhree slightly contrasted layers, has prac- and longitudinal tion similar to DDD-SH. tically no contrast. RSR-SH in longitudinal section (Fig. 5) DDD-SH in longitudinal section (Fig. 3) demonstrates a strongly positive reaction at exhibits a negative squamous region. In the
++I - I ++
-)+I-
-SS
FIG. 3. DDD-SH.
FIG
I. 4. DDD-SH.
AND
-SH
GROUPS
Longitudinal section. 400X.
Transverse section. 400X.
sub-squamous region there is a strongly positive reaction which gives rise to crescent-like stru ctures. This reaction is also positive betwec:n and below these structures up to the filan nents but not so much as in the struc-
IN
ASCARIS
CUTICLE
265
FI
:G. 5. RSR-SH.
Longitudinal section. 400X.
FI
:G. 6. RSR-SH.
Transverse section. 400X.
tres proper. The anhistous region is comPI.etely negative. Positive parts can be seen the fibrous region, strongly resembling Loseperceived with DDD-SH. RSR-SH in transverse section (Fig. 6) tt
266
CARBONELL
FIG.
7.
DDD-SS.
AND
APITZ
Longitudinal
C.
section.
600X.
negative, as are the anhistous, the fibrous and the sub-squamous regions. DDD--SS in transverse section (Fig. 8) shows a continuous positive layer in the squamous region at the periphery. The remaining regions are completely negative. None of the above regions are seen with eit,her hematoxylin-eosin or trichrome. With the use of trichrome, several layers are evidenced in the fibrous region that stain intensely positive in red. The birefringence is mild in the squamous, very strong in the sub-squamous, and negative in the anhistous region. It is also strongly positive in t,he three layers of the fibrous region. DISCUSSION
FIG.
8.
DDD-SS.
Transverse
section.
400X.
shows a negative, squamous region. Both the sub-squamous region and the filaments which stand out against the negativity of the anhistous region are strongly positive. The fibrous region is moderately posit’ive throughout, but weaker in the central layer. DDD-SS in longitudinal section (Fig. 7) shows a strongly positive react,ion in the outermost layer of the squamous region. Below this layer the react,ion is completely
In comparing our findings with the morphological description given by Hyman (1951) it is found that Hyman’s cortex corresponds to what we call the squamous and t,he sub-squamous regions; the matrix to the anhistous region and t’he basal and fibrous layers to the fibrous region and its three layers. With t)he use of hematoxylin-eosin and t,richrome a difference may be established between the three regions described by Hyman but not for those described by the authors as the present description is based upon histochemical data. A comparison of these data will clearly show how these
-SS
AND -SH
GROUPS IN ASCARIS CUTICLE
267
groups turn from -SH to -SS. In fact the The RSR-SH reaction does not coincide the cuticle in the squa-SH groups appear at first in the form of with the DDD-SH, fibrils in the anhistous region, these fibrils mous region being completely negative with becoming denser in the vicinity of the sub- RSR-SH and strongly positive with squamous region until they give a positive DDD-SH. Furthermore no positive RSRreaction in this tract which becomes strongly SH reactions appear in the crescent-like intense in the squamous region. The -SS structures. With DDD-SH, positive tracts groups appear in the squamous region alone are seen in the fibrous region. These tracts but only as successive layers, the outermost are considered to be due to the reaction of one being the most strongly positive. The the collagen, which should not be ascribed point at which both reactions are seen to to the -SH groups (Pearse, 1954) but to a coincide, with the use of DDD, is at the special affinity of the naphthol groups of the level of the squamous region. Wilkerson and reagent. Tulane (1939) found that the keratinization ACKNOWLEDGMENTS of skin into nails and hair was accompanied We wish to thank Mr. Alfred0 Pansini for transby an increase in cystine and a decrease in lating the Spanish text and Mr. H. Kabe for procmethionine. Chitwood (1938), in his studies essing the photographic prints. of the cuticle of Ascaris with the use of bioREFERENCES chemical methods, reported the presence of 35 % of matricine, which is rich in sulfur, BARRNETT, R. J., AND SELIGMAN, A. M. 1952. but did not state to what substance this Histochemical demonstration of protein sulfur was chemically bound. The -SS bound sulphydryl groups. Science 116, 323groups found in the squamous region of the 327. CHITWOOD, B. G. 1938. Observation on the chemicuticle of Ascaris seem to be in relation with cal nature of the cuticle of Ascaris. Proceedcystine, the -SH groups being apparently ings of the Helminthological Society of Washrelated to cysteine and not to methionine. ington, D. C. 3. It would be of interest to do a biochemical GOMORI, G. 1950. A rapid one step trichrome determination of the -SH, -SS and -S stain. American Journal of Clinical Pathology (methionine) groups in the cuticle of Ascaris 20. 661-664. since the transformation process of these GURR, E. 1958. Methods of Analytic Histology groups is apparently not in relation with and Histochemistry. Leonard Hill (Books) that of the skin in mammals. Limited, London. With DDD-SH, positive parts can be HYMAN, L. H. 1951. The invertebrates: Acanthocephala, Aschelminthis and Entoprocta. Vol. seen at the level of the fibrous region extendIII, McGraw-Hill Book Company, Inc., Loning throughout the three layers. It is interdon. esting to note, however, that the anhistous PEARSE, A. G. 1954. Histochemistry, Theoretical region is completely negative, the -SH and Applied. Little, Brown and Company, groups appearing only in the form of fibrils Boston. in the most peripheral tract of the above WILKERSON, V. A., AND TULANE, V. J. 1939. The mentioned region. It is believed that the chemistry of human skin. III. The occurrence -SH groups somehow combine in the anof methionine in human skin (stratum corhistous region, thus prevent,ing a reaction neum). JournaZ of Biological Chemistry 129. with DDD. 477-479.
PARASITOLOGY
Sulfhydryl
10,
263-267 (1960)
and Disulfide Groups in the Cuticle of Ascaris lurnbricoides var. suis Luis
M. Carbonell
Seccidn Medicina,
Instituto Apartado
(Submitted
and
Rafael
Apitz
Venezolano de Investigaciones 1827, Caracas, Venezuela for publication,
26 April
C. Cienti$cas,
1960)
The reactions obtained for sulfhydryl and disulfide groups in the cuticle of A.scaris Zumbricoides have demonstrated the transformation of sulfhydryls into disulfides. The -SH groups appear in the anhistous region to form a type of thin fibrils, more dense towards the periphery. The -SS and -SH groups coincide in the squamous region.
Hyman (1951), in her studies of nematodes, disclosed the presence of several regions in the cuticle of Ascaris lumbricoides: i.e. a cortex, a matrix, a fibrous and a basal layer. A study of the distribution of the sulfhydryl and disulfide groups is herein reported. No clear correlation between morphology and said distribution was found. A new histochemical description of the cuticle of Ascaris is made on the basis of the aforementioned groups. MATERIAL
AND METHODS
Specimens of A. lumbricoides var. suis, male and female, were obtained alive from the slaughterhouse, taken to the laboratory in 1% NaCl, dissected, fixed in 1% trichloroacetic acid in 80 % ethyl alcohol and embedded in vacua in paraffin. Longitudinal and transverse sections were cut at 5 p. The following reactions were used to demonstrate the -SH groups : ChevremontFrederic, RSR-SH (red sulfhydryl reagent) (Pearse, 1954) and DDD-SH (2,2’- Dihydroxy-6,6’-Dinaphtil-Disulfide) (Barrnett and Seligman, 1952). DDD-SH, inhibited with 18.6 % iodo-acetic acid at pH 8 and reactivated with 10 % potassium cyanide (Gurr, 1958) was used for the -SS group, as well as hematoxylin-eosin, Gomori’s trichrome (Gomori, 1950), phase contrast and birefringence.
RESULTS
In longitudinal section a well contrasted region, 1.6 p in thickness, is seen at the periphery under oil immersion phase contrast (Fig. 1). This region for which we have devised the name of “squamous region,” is formed by felt-like, angular-ended structures, corrugated for the most part and separated from one another by small noncontrasted interstices. The part immediately below, which we call “sub-squamous region,” is 10.6 /I in thickness and exhibits a negative contrast and a fringed appearance along its From this tract emerge internal tract. slightly contrasted filaments encroaching upon the outermost portion of the “anhistous region” which is 33-p thick. No contrast or structures are seen in this region. The fourth region, called the “fibrous region” can be divided into three layers: i.e. outer, central, and inner layer. The outer layer shows fibres in transverse section with a regular arrangement; the central layer contains zones which are contrasted in relation to the other layers described above; the inner layer shows a low contrast. The transverse sections (Fig. 2) examined under phase contrast demonstrate a continuous, highly contrasted squamous region. The sub-squamous region shows no fringes and has a spongy appearance. The anhistous region, unlike the fibrous one which has
263
264
CARBONELL
FIG. 1. This figure and all that follow are photomicrographs of the -SH and -SS groups in the cuticle of Ascaris lumbricoides var. suis. Phase contrast. Longitudinal section. 400X. A: Squamous region. B: Sub-squamous region. C: Anhistous region. D: Fibrous region.
AND
APITZ
C.
FIG. 2. Phase contrast. Transverse section. 400X. A: Squamous region. B: Sub-squamous region. C : Anhistous region. D : Fibrous region.
the periphery of the squamous region. This reaction in this region shows two positive layers, the positivity decreasing inwards till TABLE I it becomes completely negative, further reDistribution of Sulfhydryl and Disul$de Groups vealing a homogeneous, entirely negative in the Cuticle of Ascaris lumbricoides var. suis region. The sub-squamous region is perfectly recognizable but, like the filaments, is less co positive than the squamous region. The an)Y; .8 82 histous region is completely negative. PosiF4 tive portions are seen in the fibrous region, at regular intervals, all along the three laySquamous region ++ f ers. The inner layer shows a positive reaction Sub-squamous region f + ++ Anhistous region bhroughout, but not so positive as in the + Fibrous region squamous region. outer layer + ++ DDD-SH in transverse section (Fig. 4) central layer + ++ CL demonstrates a strongly positive reaction in inner layer ++ (1) the squamous region. The sub-squamous region is slightly positive and not too visible. (1) Positive in regular-banded structure throughout the layer. The anhistous region is negative, while the ++ Strongly positive. fibrous region is positive throughout but + Positive. weaker in the central layer. - Negative. Chevremont-Frederic -SH in transverse sections shows a distributhree slightly contrasted layers, has prac- and longitudinal tion similar to DDD-SH. tically no contrast. RSR-SH in longitudinal section (Fig. 5) DDD-SH in longitudinal section (Fig. 3) demonstrates a strongly positive reaction at exhibits a negative squamous region. In the
++I - I ++
-)+I-
-SS
FIG. 3. DDD-SH.
FIG
I. 4. DDD-SH.
AND
-SH
GROUPS
Longitudinal section. 400X.
Transverse section. 400X.
sub-squamous region there is a strongly positive reaction which gives rise to crescent-like stru ctures. This reaction is also positive betwec:n and below these structures up to the filan nents but not so much as in the struc-
IN
ASCARIS
CUTICLE
265
FI
:G. 5. RSR-SH.
Longitudinal section. 400X.
FI
:G. 6. RSR-SH.
Transverse section. 400X.
tres proper. The anhistous region is comPI.etely negative. Positive parts can be seen the fibrous region, strongly resembling Loseperceived with DDD-SH. RSR-SH in transverse section (Fig. 6) tt
266
CARBONELL
FIG.
7.
DDD-SS.
AND
APITZ
Longitudinal
C.
section.
600X.
negative, as are the anhistous, the fibrous and the sub-squamous regions. DDD--SS in transverse section (Fig. 8) shows a continuous positive layer in the squamous region at the periphery. The remaining regions are completely negative. None of the above regions are seen with eit,her hematoxylin-eosin or trichrome. With the use of trichrome, several layers are evidenced in the fibrous region that stain intensely positive in red. The birefringence is mild in the squamous, very strong in the sub-squamous, and negative in the anhistous region. It is also strongly positive in t,he three layers of the fibrous region. DISCUSSION
FIG.
8.
DDD-SS.
Transverse
section.
400X.
shows a negative, squamous region. Both the sub-squamous region and the filaments which stand out against the negativity of the anhistous region are strongly positive. The fibrous region is moderately posit’ive throughout, but weaker in the central layer. DDD-SS in longitudinal section (Fig. 7) shows a strongly positive react,ion in the outermost layer of the squamous region. Below this layer the react,ion is completely
In comparing our findings with the morphological description given by Hyman (1951) it is found that Hyman’s cortex corresponds to what we call the squamous and t,he sub-squamous regions; the matrix to the anhistous region and t’he basal and fibrous layers to the fibrous region and its three layers. With t)he use of hematoxylin-eosin and t,richrome a difference may be established between the three regions described by Hyman but not for those described by the authors as the present description is based upon histochemical data. A comparison of these data will clearly show how these
-SS
AND -SH
GROUPS IN ASCARIS CUTICLE
267
groups turn from -SH to -SS. In fact the The RSR-SH reaction does not coincide the cuticle in the squa-SH groups appear at first in the form of with the DDD-SH, fibrils in the anhistous region, these fibrils mous region being completely negative with becoming denser in the vicinity of the sub- RSR-SH and strongly positive with squamous region until they give a positive DDD-SH. Furthermore no positive RSRreaction in this tract which becomes strongly SH reactions appear in the crescent-like intense in the squamous region. The -SS structures. With DDD-SH, positive tracts groups appear in the squamous region alone are seen in the fibrous region. These tracts but only as successive layers, the outermost are considered to be due to the reaction of one being the most strongly positive. The the collagen, which should not be ascribed point at which both reactions are seen to to the -SH groups (Pearse, 1954) but to a coincide, with the use of DDD, is at the special affinity of the naphthol groups of the level of the squamous region. Wilkerson and reagent. Tulane (1939) found that the keratinization ACKNOWLEDGMENTS of skin into nails and hair was accompanied We wish to thank Mr. Alfred0 Pansini for transby an increase in cystine and a decrease in lating the Spanish text and Mr. H. Kabe for procmethionine. Chitwood (1938), in his studies essing the photographic prints. of the cuticle of Ascaris with the use of bioREFERENCES chemical methods, reported the presence of 35 % of matricine, which is rich in sulfur, BARRNETT, R. J., AND SELIGMAN, A. M. 1952. but did not state to what substance this Histochemical demonstration of protein sulfur was chemically bound. The -SS bound sulphydryl groups. Science 116, 323groups found in the squamous region of the 327. CHITWOOD, B. G. 1938. Observation on the chemicuticle of Ascaris seem to be in relation with cal nature of the cuticle of Ascaris. Proceedcystine, the -SH groups being apparently ings of the Helminthological Society of Washrelated to cysteine and not to methionine. ington, D. C. 3. It would be of interest to do a biochemical GOMORI, G. 1950. A rapid one step trichrome determination of the -SH, -SS and -S stain. American Journal of Clinical Pathology (methionine) groups in the cuticle of Ascaris 20. 661-664. since the transformation process of these GURR, E. 1958. Methods of Analytic Histology groups is apparently not in relation with and Histochemistry. Leonard Hill (Books) that of the skin in mammals. Limited, London. With DDD-SH, positive parts can be HYMAN, L. H. 1951. The invertebrates: Acanthocephala, Aschelminthis and Entoprocta. Vol. seen at the level of the fibrous region extendIII, McGraw-Hill Book Company, Inc., Loning throughout the three layers. It is interdon. esting to note, however, that the anhistous PEARSE, A. G. 1954. Histochemistry, Theoretical region is completely negative, the -SH and Applied. Little, Brown and Company, groups appearing only in the form of fibrils Boston. in the most peripheral tract of the above WILKERSON, V. A., AND TULANE, V. J. 1939. The mentioned region. It is believed that the chemistry of human skin. III. The occurrence -SH groups somehow combine in the anof methionine in human skin (stratum corhistous region, thus prevent,ing a reaction neum). JournaZ of Biological Chemistry 129. with DDD. 477-479.