Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, 2016
Glycine (Gly) substitutions in collagen Gly-X-Y repeats disrupt folding of type I procollagen tri... more Glycine (Gly) substitutions in collagen Gly-X-Y repeats disrupt folding of type I procollagen triple helix and cause severe bone fragility and malformations (osteogenesis imperfecta, aka OI). However, these mutations do not elicit the expected Endoplasmic Reticulum (ER) stress response, in contrast to other protein folding diseases. Thus, it has remained unclear whether cell stress and osteoblast malfunction contribute to the bone pathology caused by Gly substitutions. Here we used a mouse with a Gly610 to cysteine (Cys) substitution in the procollagen α2(I) chain to show that misfolded procollagen accumulation in the ER leads to an unusual form of cell stress, which is neither a conventional unfolded protein response stress nor ER overload. Despite pronounced ER dilation, there is no upregulation of BIP expected in the former and no activation of NFκB signaling expected in the latter. Altered expression of ER chaperones αB crystalline and HSP47, phosphorylation of EIF2α, activation...
Lyme disease (borreliosis) incidence continues to increase despite a growing knowledge of primary... more Lyme disease (borreliosis) incidence continues to increase despite a growing knowledge of primary and secondary prevention strategies. Primary prevention aims to reduce the risk of tick exposure and thereby decrease the incidence of new Lyme disease cases. Secondary prevention targets the development of disease or reduces disease severity among people who have been bitten by infected ticks. Numerous prevention strategies are available, and although they vary in cost, acceptability and effectiveness, uptake has been universally poor. Research in areas where Lyme disease is endemic has demonstrated that despite adequate knowledge about its symptoms and transmission, many people do not perform behaviors to reduce their risk of infection. New prevention strategies should aim to increase people's confidence in their ability to carry out preventive behaviors, raise awareness of desirable outcomes, and aid in the realization that the necessary skills and resources are available for preventive measures to be taken. In this article we evaluate the prevention and treatment strategies for Lyme disease, and discuss how these strategies can be implemented effectively. As many patients with Lyme disease develop arthritis and are referred to rheumatologists it is important that these health-care providers can educate patients about disease-prevention strategies.
FSH, the primary trophic hormone for gamete development in mammals, is composed of two protein su... more FSH, the primary trophic hormone for gamete development in mammals, is composed of two protein subunits, alpha and beta. It is known that 17 beta-estradiol (E2) and progesterone (P4) can decrease the secretion and synthesis of FSH in ovine pituitary cultures. Data presented here indicate that E2 and P4 decrease the steady state levels of FSH beta mRNA concomitantly with FSH secretion in ovine pituitary cultures. By 24 h, E2 decreased the steady state levels of FSH beta mRNA and FSH secretion by 68% +/- 5%. P4 also decreased both concomitantly, but by 58% +/- 7% after 24 h. E2 and P4 also decrease steady state levels of alpha mRNA, but at a lower rate. Finally, it is shown that E2 and P4 decrease transcription of the FSH beta by greater than 85% in 2 h; alpha mRNA transcription is decreased by 70% in 12 h. These effects are not altered even when cycloheximide is present to block protein synthesis by 95%. These data further define the mechanisms whereby E2 and P4 inhibit ovine FSH secretion/synthesis directly at the pituitary level. They also provide the first example of negative transcriptional regulation by P4 and the second of two examples now established for E2.
The specific mechanisms of collagen induction in human dermal fibroblasts by ascorbic acid and tr... more The specific mechanisms of collagen induction in human dermal fibroblasts by ascorbic acid and transforming growth factor-beta 1 (TGF-beta 1) and their effect in combination are uncertain. Collagen synthesis and steady-state levels of pro alpha 1(I) and pro alpha 1(III) collagen RNA were examined in human dermal fibroblasts treated with 100 microM ascorbic acid, 2.5 ng/ml TGF-beta 1, or both. Within 72 h ascorbic acid and TGF-beta 1 had increased collagen synthesis by 2.55 +/- 0.32- and 1.98 +/- 0.13-fold, respectively; in the presence of both, collagen synthesis increased 4.51 +/- 0.74-fold, appearing additive. Ascorbic acid acts specifically by increasing relative collagen synthesis whereas TGF-beta 1 increases overall protein synthesis. Steady-state levels of the pro alpha 1(I) collagen (5.8 and 4.8 kb) and pro alpha 1(III) collagen (5.4 and 4.8 kb) mRNAs were examined independently. Under each condition the steady-state levels of the longer transcripts for pro alpha 1(I) and pro alpha 1(III) collagens appeared coordinately and preferentially elevated. In the presence of both ascorbic acid and TGF-beta 1 the steady-state RNA levels did not increase in an additive manner, suggesting that the additive increase in collagen synthesis results from additional post-transcriptional mechanisms.
In the field of biomechanics, collagen fibrils are believed to be robust mechanical structures ch... more In the field of biomechanics, collagen fibrils are believed to be robust mechanical structures characterized by a low extensibility. Until very recently, information on the mechanical properties of collagen fibrils could only be derived from ensemble measurements performed on complete tissues such as bone, skin and tendon. Here we measure force-elongation/relaxation profiles of single collagen fibrils using atomic force microscopy-based force spectroscopy. The elongation profiles indicate that in vitro assembled heterotrimeric type I collagen fibrils are characterized by a large extensibility. Numerous discontinuities and a plateau in the force profile indicate major reorganization occurs within the fibrils in the 1.5 -- 4.5 nN range. Our study demonstrates that newly assembled collagen fibrils are robust structures with a significant reserve of elasticity that could play a determinant role in cellular motion in the context of tissue growth and morphogenesis. In contrast, homotrimer...
Null alleles for the COL5A1 gene and missense mutations for COL5A1 or the COL5A2 gene underlie ca... more Null alleles for the COL5A1 gene and missense mutations for COL5A1 or the COL5A2 gene underlie cases of classic Ehlers-Danlos syndrome, characterized by fragile, hyperextensible skin and hypermobile joints. However, no classic Ehlers-Danlos syndrome case has yet been associated with COL5A2 null alleles, and phenotypes that might result from such alleles are unknown. We describe mice with null alleles for the Col5a2. Col5a2(-/-) homozygosity is embryonic lethal at approximately 12 days post conception. Unlike previously described mice null for Col5a1, which die at 10.5 days post conception and virtually lack collagen fibrils, Col5a2(-/-) embryos have readily detectable collagen fibrils, thicker than in wild-type controls. Differences in Col5a2(-/-) and Col5a1(-/-) fibril formation and embryonic survival suggest that α1(V)3 homotrimers, a rare collagen V isoform that occurs in the absence of sufficient levels of α2(V) chains, serve functional roles that partially compensate for loss o...
Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, Jan 31, 2015
Osteogenesis imperfecta (OI) is a heterogenous heritable connective tissue disorder associated wi... more Osteogenesis imperfecta (OI) is a heterogenous heritable connective tissue disorder associated with reduced bone mineral density and skeletal fragility. Bone is inherently mechanosensitive, with bone strength being proportional to muscle mass and strength. Physically active healthy children accrue more bone than inactive children. Children with type I OI exhibit decreased exercise capacity and muscle strength compared to healthy peers. It is unknown whether this muscle weakness reflects decreased physical activity or a muscle pathology. In the following study, we used heterozygous G610C OI model mice (+/G610C), which model both the genotype and phenotype of a large Amish OI kindred, to evaluate hindlimb muscle function and physical activity levels prior to evaluating the ability of +/G610C mice to undergo a treadmill exercise regimen. We found +/G610C mice hindlimb muscles do not exhibit compromised muscle function and their activity levels were not reduced relative to wildtype mice...
The turnaround time for diagnosis of maple syrup urine disease (MSUD) by classic serum amino acid... more The turnaround time for diagnosis of maple syrup urine disease (MSUD) by classic serum amino acid analyses often requires 3-4 days. This is due to the need for branched-chain amino acids (BCAA) to accumulate in the serum of the newborn before testing. The accumulation of BCAAs in infants with MSUD during this time increases the risk of the infant becoming clinically symptomatic. We have developed a noninvasive DNA-based mismatch PCR-RFLP assay for the Y393N BCKDHA allele (E1alpha gene of the branched chain alpha-keto acid dehydrogenase complex), the primary cause of MSUD in Old Order Mennonite communities. The homozygosity and high frequency of this mutation in the Mennonite community and its prevalence in compound heterozygote non-Mennonite MSUD patients is of significance. We describe carrier testing, present the results of nine newborns diagnostically evaluated for the Y393N BCKDHA allele, and demonstrate the efficacy of this PCR-RFLP assay for determining clinical status within ...
Short measures of health status are used increasingly in health services research, yet their sens... more Short measures of health status are used increasingly in health services research, yet their sensitivities to clinical change have not been compared with longer, established instruments. In this study, 5 health status measures were administered preoperatively and 3 months postoperatively to 54 patients undergoing total hip arthroplasty. These instruments included the Sickness Impact Profile (SIP)--an established, long measure--and 4 short forms: the SF-36, Functional Status Questionnaire, shortened Arthritis Impact Measurement Scales, and Modified Health Assessment Questionnaire. Scores for physical, psychological, and global dimensions were constructed by aggregating subscales. Sensitivity to change, or responsiveness, was expressed with the standardized response mean (SRM), calculated as the mean change in score divided by the standard deviation of the change in score. The sampling distribution of the SRM was estimated with a jackknife procedure. Preoperative scores were moderately to highly correlated across instruments. The physical and global dimension SRMs of the brief health status measures ranged from 0.85 to 1.27 and were as large as or larger than the corresponding SIP SRMs. The SIP had the highest SRM on the psychological dimension. None of the instruments was significantly more sensitive than the others at the critical value (P = 0.005) adjusted for multiple comparisons. The brief health status measures were equally or more responsive than the SIP after total hip arthroplasty in the physical and global dimensions. Much larger samples are required to demonstrate statistically significant differences in SRMs among instruments.
Lyme disease (borreliosis) incidence continues to increase despite a growing knowledge of primary... more Lyme disease (borreliosis) incidence continues to increase despite a growing knowledge of primary and secondary prevention strategies. Primary prevention aims to reduce the risk of tick exposure and thereby decrease the incidence of new Lyme disease cases. Secondary prevention targets the development of disease or reduces disease severity among people who have been bitten by infected ticks. Numerous prevention strategies are available, and although they vary in cost, acceptability and effectiveness, uptake has been universally poor. Research in areas where Lyme disease is endemic has demonstrated that despite adequate knowledge about its symptoms and transmission, many people do not perform behaviors to reduce their risk of infection. New prevention strategies should aim to increase people's confidence in their ability to carry out preventive behaviors, raise awareness of desirable outcomes, and aid in the realization that the necessary skills and resources are available for preventive measures to be taken. In this article we evaluate the prevention and treatment strategies for Lyme disease, and discuss how these strategies can be implemented effectively. As many patients with Lyme disease develop arthritis and are referred to rheumatologists it is important that these health-care providers can educate patients about disease-prevention strategies.
FSH, the primary trophic hormone for gamete development in mammals, is composed of two protein su... more FSH, the primary trophic hormone for gamete development in mammals, is composed of two protein subunits, and β. It is known that 17β-estradiol (E 2 ) and progesterone (P 4 ) can decrease the secretion and synthesis of FSH in ovine pituitary cultures. Data presented here ...
Quality of life assessment has become central to health services research. Many reliable, valid m... more Quality of life assessment has become central to health services research. Many reliable, valid methods are available to evaluate quality of life19 including measures of functional status, categorical rating scales in which patients choose a single number to represent ...
Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, 2016
Glycine (Gly) substitutions in collagen Gly-X-Y repeats disrupt folding of type I procollagen tri... more Glycine (Gly) substitutions in collagen Gly-X-Y repeats disrupt folding of type I procollagen triple helix and cause severe bone fragility and malformations (osteogenesis imperfecta, aka OI). However, these mutations do not elicit the expected Endoplasmic Reticulum (ER) stress response, in contrast to other protein folding diseases. Thus, it has remained unclear whether cell stress and osteoblast malfunction contribute to the bone pathology caused by Gly substitutions. Here we used a mouse with a Gly610 to cysteine (Cys) substitution in the procollagen α2(I) chain to show that misfolded procollagen accumulation in the ER leads to an unusual form of cell stress, which is neither a conventional unfolded protein response stress nor ER overload. Despite pronounced ER dilation, there is no upregulation of BIP expected in the former and no activation of NFκB signaling expected in the latter. Altered expression of ER chaperones αB crystalline and HSP47, phosphorylation of EIF2α, activation...
Lyme disease (borreliosis) incidence continues to increase despite a growing knowledge of primary... more Lyme disease (borreliosis) incidence continues to increase despite a growing knowledge of primary and secondary prevention strategies. Primary prevention aims to reduce the risk of tick exposure and thereby decrease the incidence of new Lyme disease cases. Secondary prevention targets the development of disease or reduces disease severity among people who have been bitten by infected ticks. Numerous prevention strategies are available, and although they vary in cost, acceptability and effectiveness, uptake has been universally poor. Research in areas where Lyme disease is endemic has demonstrated that despite adequate knowledge about its symptoms and transmission, many people do not perform behaviors to reduce their risk of infection. New prevention strategies should aim to increase people's confidence in their ability to carry out preventive behaviors, raise awareness of desirable outcomes, and aid in the realization that the necessary skills and resources are available for preventive measures to be taken. In this article we evaluate the prevention and treatment strategies for Lyme disease, and discuss how these strategies can be implemented effectively. As many patients with Lyme disease develop arthritis and are referred to rheumatologists it is important that these health-care providers can educate patients about disease-prevention strategies.
FSH, the primary trophic hormone for gamete development in mammals, is composed of two protein su... more FSH, the primary trophic hormone for gamete development in mammals, is composed of two protein subunits, alpha and beta. It is known that 17 beta-estradiol (E2) and progesterone (P4) can decrease the secretion and synthesis of FSH in ovine pituitary cultures. Data presented here indicate that E2 and P4 decrease the steady state levels of FSH beta mRNA concomitantly with FSH secretion in ovine pituitary cultures. By 24 h, E2 decreased the steady state levels of FSH beta mRNA and FSH secretion by 68% +/- 5%. P4 also decreased both concomitantly, but by 58% +/- 7% after 24 h. E2 and P4 also decrease steady state levels of alpha mRNA, but at a lower rate. Finally, it is shown that E2 and P4 decrease transcription of the FSH beta by greater than 85% in 2 h; alpha mRNA transcription is decreased by 70% in 12 h. These effects are not altered even when cycloheximide is present to block protein synthesis by 95%. These data further define the mechanisms whereby E2 and P4 inhibit ovine FSH secretion/synthesis directly at the pituitary level. They also provide the first example of negative transcriptional regulation by P4 and the second of two examples now established for E2.
The specific mechanisms of collagen induction in human dermal fibroblasts by ascorbic acid and tr... more The specific mechanisms of collagen induction in human dermal fibroblasts by ascorbic acid and transforming growth factor-beta 1 (TGF-beta 1) and their effect in combination are uncertain. Collagen synthesis and steady-state levels of pro alpha 1(I) and pro alpha 1(III) collagen RNA were examined in human dermal fibroblasts treated with 100 microM ascorbic acid, 2.5 ng/ml TGF-beta 1, or both. Within 72 h ascorbic acid and TGF-beta 1 had increased collagen synthesis by 2.55 +/- 0.32- and 1.98 +/- 0.13-fold, respectively; in the presence of both, collagen synthesis increased 4.51 +/- 0.74-fold, appearing additive. Ascorbic acid acts specifically by increasing relative collagen synthesis whereas TGF-beta 1 increases overall protein synthesis. Steady-state levels of the pro alpha 1(I) collagen (5.8 and 4.8 kb) and pro alpha 1(III) collagen (5.4 and 4.8 kb) mRNAs were examined independently. Under each condition the steady-state levels of the longer transcripts for pro alpha 1(I) and pro alpha 1(III) collagens appeared coordinately and preferentially elevated. In the presence of both ascorbic acid and TGF-beta 1 the steady-state RNA levels did not increase in an additive manner, suggesting that the additive increase in collagen synthesis results from additional post-transcriptional mechanisms.
In the field of biomechanics, collagen fibrils are believed to be robust mechanical structures ch... more In the field of biomechanics, collagen fibrils are believed to be robust mechanical structures characterized by a low extensibility. Until very recently, information on the mechanical properties of collagen fibrils could only be derived from ensemble measurements performed on complete tissues such as bone, skin and tendon. Here we measure force-elongation/relaxation profiles of single collagen fibrils using atomic force microscopy-based force spectroscopy. The elongation profiles indicate that in vitro assembled heterotrimeric type I collagen fibrils are characterized by a large extensibility. Numerous discontinuities and a plateau in the force profile indicate major reorganization occurs within the fibrils in the 1.5 -- 4.5 nN range. Our study demonstrates that newly assembled collagen fibrils are robust structures with a significant reserve of elasticity that could play a determinant role in cellular motion in the context of tissue growth and morphogenesis. In contrast, homotrimer...
Null alleles for the COL5A1 gene and missense mutations for COL5A1 or the COL5A2 gene underlie ca... more Null alleles for the COL5A1 gene and missense mutations for COL5A1 or the COL5A2 gene underlie cases of classic Ehlers-Danlos syndrome, characterized by fragile, hyperextensible skin and hypermobile joints. However, no classic Ehlers-Danlos syndrome case has yet been associated with COL5A2 null alleles, and phenotypes that might result from such alleles are unknown. We describe mice with null alleles for the Col5a2. Col5a2(-/-) homozygosity is embryonic lethal at approximately 12 days post conception. Unlike previously described mice null for Col5a1, which die at 10.5 days post conception and virtually lack collagen fibrils, Col5a2(-/-) embryos have readily detectable collagen fibrils, thicker than in wild-type controls. Differences in Col5a2(-/-) and Col5a1(-/-) fibril formation and embryonic survival suggest that α1(V)3 homotrimers, a rare collagen V isoform that occurs in the absence of sufficient levels of α2(V) chains, serve functional roles that partially compensate for loss o...
Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, Jan 31, 2015
Osteogenesis imperfecta (OI) is a heterogenous heritable connective tissue disorder associated wi... more Osteogenesis imperfecta (OI) is a heterogenous heritable connective tissue disorder associated with reduced bone mineral density and skeletal fragility. Bone is inherently mechanosensitive, with bone strength being proportional to muscle mass and strength. Physically active healthy children accrue more bone than inactive children. Children with type I OI exhibit decreased exercise capacity and muscle strength compared to healthy peers. It is unknown whether this muscle weakness reflects decreased physical activity or a muscle pathology. In the following study, we used heterozygous G610C OI model mice (+/G610C), which model both the genotype and phenotype of a large Amish OI kindred, to evaluate hindlimb muscle function and physical activity levels prior to evaluating the ability of +/G610C mice to undergo a treadmill exercise regimen. We found +/G610C mice hindlimb muscles do not exhibit compromised muscle function and their activity levels were not reduced relative to wildtype mice...
The turnaround time for diagnosis of maple syrup urine disease (MSUD) by classic serum amino acid... more The turnaround time for diagnosis of maple syrup urine disease (MSUD) by classic serum amino acid analyses often requires 3-4 days. This is due to the need for branched-chain amino acids (BCAA) to accumulate in the serum of the newborn before testing. The accumulation of BCAAs in infants with MSUD during this time increases the risk of the infant becoming clinically symptomatic. We have developed a noninvasive DNA-based mismatch PCR-RFLP assay for the Y393N BCKDHA allele (E1alpha gene of the branched chain alpha-keto acid dehydrogenase complex), the primary cause of MSUD in Old Order Mennonite communities. The homozygosity and high frequency of this mutation in the Mennonite community and its prevalence in compound heterozygote non-Mennonite MSUD patients is of significance. We describe carrier testing, present the results of nine newborns diagnostically evaluated for the Y393N BCKDHA allele, and demonstrate the efficacy of this PCR-RFLP assay for determining clinical status within ...
Short measures of health status are used increasingly in health services research, yet their sens... more Short measures of health status are used increasingly in health services research, yet their sensitivities to clinical change have not been compared with longer, established instruments. In this study, 5 health status measures were administered preoperatively and 3 months postoperatively to 54 patients undergoing total hip arthroplasty. These instruments included the Sickness Impact Profile (SIP)--an established, long measure--and 4 short forms: the SF-36, Functional Status Questionnaire, shortened Arthritis Impact Measurement Scales, and Modified Health Assessment Questionnaire. Scores for physical, psychological, and global dimensions were constructed by aggregating subscales. Sensitivity to change, or responsiveness, was expressed with the standardized response mean (SRM), calculated as the mean change in score divided by the standard deviation of the change in score. The sampling distribution of the SRM was estimated with a jackknife procedure. Preoperative scores were moderately to highly correlated across instruments. The physical and global dimension SRMs of the brief health status measures ranged from 0.85 to 1.27 and were as large as or larger than the corresponding SIP SRMs. The SIP had the highest SRM on the psychological dimension. None of the instruments was significantly more sensitive than the others at the critical value (P = 0.005) adjusted for multiple comparisons. The brief health status measures were equally or more responsive than the SIP after total hip arthroplasty in the physical and global dimensions. Much larger samples are required to demonstrate statistically significant differences in SRMs among instruments.
Lyme disease (borreliosis) incidence continues to increase despite a growing knowledge of primary... more Lyme disease (borreliosis) incidence continues to increase despite a growing knowledge of primary and secondary prevention strategies. Primary prevention aims to reduce the risk of tick exposure and thereby decrease the incidence of new Lyme disease cases. Secondary prevention targets the development of disease or reduces disease severity among people who have been bitten by infected ticks. Numerous prevention strategies are available, and although they vary in cost, acceptability and effectiveness, uptake has been universally poor. Research in areas where Lyme disease is endemic has demonstrated that despite adequate knowledge about its symptoms and transmission, many people do not perform behaviors to reduce their risk of infection. New prevention strategies should aim to increase people's confidence in their ability to carry out preventive behaviors, raise awareness of desirable outcomes, and aid in the realization that the necessary skills and resources are available for preventive measures to be taken. In this article we evaluate the prevention and treatment strategies for Lyme disease, and discuss how these strategies can be implemented effectively. As many patients with Lyme disease develop arthritis and are referred to rheumatologists it is important that these health-care providers can educate patients about disease-prevention strategies.
FSH, the primary trophic hormone for gamete development in mammals, is composed of two protein su... more FSH, the primary trophic hormone for gamete development in mammals, is composed of two protein subunits, and β. It is known that 17β-estradiol (E 2 ) and progesterone (P 4 ) can decrease the secretion and synthesis of FSH in ovine pituitary cultures. Data presented here ...
Quality of life assessment has become central to health services research. Many reliable, valid m... more Quality of life assessment has become central to health services research. Many reliable, valid methods are available to evaluate quality of life19 including measures of functional status, categorical rating scales in which patients choose a single number to represent ...
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Papers by Charlotte Phillips