Molly is a 10-year-old girl of short stature with hyperelastic skin which bruises easily, and hypermobile joints which are easily dislocated. Molly's mother said that Molly's diet includes fruits and vegetables high in Vitamin C, so she does not have a vitamin C deficiency. She also has no signs of anemia and has normal iron levels. You decide to refer Molly to a surgeon who will correct the looseness of Molly's joints. The surgeon agrees to take skin samples during the surgery so that you can perform an analysis of her fibroblasts. These cells produce connective tissue in the skin. Because skin, ligaments, and tendons have collagen as their major structural protein, you suspect that Molly is unable to synthesize collagen properly and you wish to test this hypothesis. That evening, you have dinner with your old college friend, who is now a veterinarian. You describe Molly's symptoms to her and she remarks that Molly's symptoms sound remarkably similar to a condition called dermatosparaxis (literally “torn skin") that she has observed in sheep and cattle. She agrees with you that the disorder probably is due to improper collagen formation, and generously offers a sample of collagen from a dermatosparactic sheep in her clinic that you can use in your experiments. You are able to obtain fibroblasts from the samples of Molly's skin and the skin from the dermatosparactic sheep, as well as normal control fibroblasts capable of secreting normal collagen You carry out SDS-PAGE analysis of the collagen extracted from a normal control, patient, and sheep fibroblasts. The results are shown in the image attached. You observe that there is an extra band in Molly's collagen that has a slightly higher molecular weight than the molecular weight of the normal control sample α2 chain. In the sheep sample, there are two bands with molecular weights greater than both normal control sample α1(I) and α2 chains. The increase in size for collagen bands, suggests that there is an issue with the cleavage step in synthesis. However, the cause for that change is not able to be determined by the SDS-PAGE gel alone, and appears to be different for patient Molly and the sheep. Figure 2 (attached). SDS-PAGE analysis of collagen isolated from normal control, patient, and sheep fibroblasts. The ratios at the bottom of the gel are the ratio of the intensities of the bands (from top to bottom) as determined by densiometric scanning. Larger proteins are at the top of the gel, smaller proteins migrate faster to the bottom. Following up on the SDS-PAGE results above, you carry out a series of experiments outlined in Tables 1, 2 and 3. In the first set of experiments, the cultured fibroblasts are assayed for levels of key enzymes necessary for proper collagen synthesis. In the second set of experiments, an amino acid analysis of control and patient collagen is carried out. In the third set of experiments, exogenous normal enzymes are added to the collagen from the cultured fibroblast medium to see if these enzymes can correct the defect. The results are shown in the tables. Table 1: Assays of enzyme activity: Molly's fibroblast enzyme activity Dermatosparactic sheep fibroblast enzyme activity Procollagen N-peptidase Normal Low Procollagen C-peptidase Normal Normal Prolyl hydroxylase Normal Normal Lysyl hydroxylase Normal Normal Table 2: Relative amino acid compositions: Collagen extracted from Molly's fibroblasts Collagen extracted from dermatosparactic sheep 4-Hydroxyproline ( 4-Hyp) Normal Normal 3-Hydroxyproline (3-Hyp) Normal Normal Hydroxylysine (Hyl) Normal Normal Table 3: Incubation of collagen with exogenous enzyme from a normal source: Collagen extracted from Molly's fibroblasts Collagen extracted from dermatosparactic sheep Procollagen N-peptidase No change SDS-PAGE results show collagen is the same as the control after digestion. Procollagen C-peptidase No change No change Tables 1, 2, and 3: Experiments with collagen taken from Molly, a patient with a collagen defect and a dermatosparactic sheep. Do you think the defect in collagen synthesis in Molly's fibroblasts is different from the defect in the dermatosparactic sheep? If you use outside scientific literature sources, be sure to cite them.
Molly is a 10-year-old girl of short stature with hyperelastic skin which bruises easily, and hypermobile joints which are easily dislocated. Molly's mother said that Molly's diet includes fruits and vegetables high in Vitamin C, so she does not have a vitamin C deficiency. She also has no signs of anemia and has normal iron levels.
You decide to refer Molly to a surgeon who will correct the looseness of Molly's joints. The surgeon agrees to take skin samples during the surgery so that you can perform an analysis of her fibroblasts. These cells produce connective tissue in the skin. Because skin, ligaments, and tendons have collagen as their major structural protein, you suspect that Molly is unable to synthesize collagen properly and you wish to test this hypothesis.
That evening, you have dinner with your old college friend, who is now a veterinarian. You describe Molly's symptoms to her and she remarks that Molly's symptoms sound remarkably similar to a condition called dermatosparaxis (literally “torn skin") that she has observed in sheep and cattle. She agrees with you that the disorder probably is due to improper collagen formation, and generously offers a sample of collagen from a dermatosparactic sheep in her clinic that you can use in your experiments. You are able to obtain fibroblasts from the samples of Molly's skin and the skin from the dermatosparactic sheep, as well as normal control fibroblasts capable of secreting normal collagen
You carry out SDS-PAGE analysis of the collagen extracted from a normal control, patient, and sheep fibroblasts. The results are shown in the image attached. You observe that there is an extra band in Molly's collagen that has a slightly higher molecular weight than the molecular weight of the normal control sample α2 chain. In the sheep sample, there are two bands with molecular weights greater than both normal control sample α1(I) and α2 chains.
The increase in size for collagen bands, suggests that there is an issue with the cleavage step in synthesis. However, the cause for that change is not able to be determined by the SDS-PAGE gel alone, and appears to be different for patient Molly and the sheep.
Figure 2 (attached). SDS-PAGE analysis of collagen isolated from normal control, patient, and sheep fibroblasts. The ratios at the bottom of the gel are the ratio of the intensities of the bands (from top to bottom) as determined by densiometric scanning. Larger proteins are at the top of the gel, smaller proteins migrate faster to the bottom.
Following up on the SDS-PAGE results above, you carry out a series of experiments outlined in Tables 1, 2 and 3. In the first set of experiments, the cultured fibroblasts are assayed for levels of key enzymes necessary for proper collagen synthesis. In the second set of experiments, an amino acid analysis of control and patient collagen is carried out. In the third set of experiments, exogenous normal enzymes are added to the collagen from the cultured fibroblast medium to see if these enzymes can correct the defect. The results are shown in the tables.
Table 1: Assays of enzyme activity:
|
|
Molly's fibroblast enzyme activity |
Dermatosparactic sheep fibroblast enzyme activity |
|
Procollagen N-peptidase |
Normal |
Low |
|
Procollagen C-peptidase |
Normal |
Normal |
|
Prolyl hydroxylase |
Normal |
Normal |
|
Lysyl hydroxylase |
Normal |
Normal |
Table 2: Relative amino acid compositions:
|
|
Collagen extracted from Molly's fibroblasts |
Collagen extracted from dermatosparactic sheep |
|
4-Hydroxyproline ( 4-Hyp) |
Normal |
Normal |
|
3-Hydroxyproline (3-Hyp) |
Normal |
Normal |
|
Hydroxylysine (Hyl) |
Normal |
Normal |
Table 3: Incubation of collagen with exogenous enzyme from a normal source:
|
|
Collagen extracted from Molly's fibroblasts |
Collagen extracted from dermatosparactic sheep |
|
Procollagen N-peptidase |
No change |
SDS-PAGE results show collagen is the same as the control after digestion. |
|
Procollagen C-peptidase |
No change |
No change |
Tables 1, 2, and 3: Experiments with collagen taken from Molly, a patient with a collagen defect and a dermatosparactic sheep.
Do you think the defect in collagen synthesis in Molly's fibroblasts is different from the defect in the dermatosparactic sheep? If you use outside scientific literature sources, be sure to cite them.
1. Amount of Vitamin C in Jane’s diet fails to improve Jane’s symptoms because the dermatosparaxis occurs due to the problem in the collagen molecule which is important in connective tissue of skin, ligaments, and tendons.
So vitamin C has no effect here.
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