Hydroxylation Of Proline In Collagen Research Articles

Bioengineered vascular grafts with a pathogenic TGFBR1 variant model aneurysm formation in vivo and reveal underlying collagen defects.

Thoracic aortic aneurysm (TAA) is a life-threatening vascular disease frequently associated with underlying genetic causes. An inadequate understanding of human TAA pathogenesis highlights the need for better disease models. Here, we established a functional human TAA model in an animal host by combining human induced pluripotent stem cells (hiPSCs), bioengineered vascular grafts (BVGs), and gene editing. We generated BVGs from isogenic control hiPSC-derived vascular smooth muscle cells (SMCs) and mutant SMCs gene-edited to carry a Loeys-Dietz syndrome (LDS)-associated pathogenic variant (TGFBR1A230T). We also generated hiPSC-derived BVGs using cells from a patient with LDS (PatientA230T/+) and using genetically corrected cells (Patient+/+). Control and experimental BVGs were then implanted into the common carotid arteries of nude rats. The TGFBR1A230T variant led to impaired mechanical properties of BVGs, resulting in lower burst pressure and suture retention strength. BVGs carrying the variant dilated over time in vivo, resembling human TAA formation. Spatial transcriptomics profiling revealed defective expression of extracellular matrix (ECM) formation genes in PatientA230T/+ BVGs compared with Patient+/+ BVGs. Histological analysis and protein assays validated quantitative and qualitative ECM defects in PatientA230T/+ BVGs and patient tissue, including decreased collagen hydroxylation. SMC organization was also impaired in PatientA230T/+ BVGs as confirmed by vascular contraction testing. Silencing of collagen-modifying enzymes with small interfering RNAs reduced collagen proline hydroxylation in SMC-derived tissue constructs. These studies demonstrated the utility of BVGs to model human TAA formation in an animal host and highlighted the role of reduced collagen modifying enzyme activity in human TAA formation.

Cycloheximide promotes type I collagen maturation mainly via collagen prolyl 4-hydroxylase subunit α2.

Aberrant deposition of collagen is associated with cancer development and tissue fibrosis. Proline hydroxylation, catalyzed by collagen prolyl 4-hydroxylases (C-P4Hs), is necessary for collagen maturation and secretion. Here, we try to evaluate the mechanism of the regulation of CHX on collagen maturation. Using pepsin digestion, liquid chromatograph mass spectrometry and gene knockout, we find that treatment of mouse embryonic fibroblasts with cycloheximide (CHX) increases type I collagen proline hydroxylation partially via P4HA1 and mainly via P4HA2. Western blot analysis results show that CHX treatment reduces type I collagen but does not obviously impact the level of P4HA1/2 protein in the endoplasmic reticulum, which enhances the molar ratio of P4HA1/2 to type I collagen, and coimmunoprecipitation results confirm that more P4HA1/2 can bind to each type I collagen. Since C-P4Hs possess the capability to hydroxylate proline independent of ascorbate for a few cycles, this enhanced binding between P4HA1/2 and type I collagen can partially explain how CHX stimulates type I collagen maturation.

Cotranslational prolyl hydroxylation is essential for flavivirus biogenesis.

Viral pathogens are an ongoing threat to public health worldwide. Analysing their dependence on host biosynthetic pathways could lead to effective antiviral therapies1. Here we integrate proteomic analyses of polysomes with functional genomics and pharmacological interventions to define how enteroviruses and flaviviruses remodel host polysomes to synthesize viral proteins and disable host protein production. We find that infection with polio, dengue or Zika virus markedly modifies polysome composition, without major changes to core ribosome stoichiometry. These viruses use different strategies to evict a common set of translation initiation and RNA surveillance factors from polysomes while recruiting host machineries that are specifically required for viral biogenesis. Targeting these specialized viral polysomes could provide a new approach for antiviral interventions. For example, we find that both Zika and dengue use the collagen proline hydroxylation machinery to mediate cotranslational modification of conserved proline residues in the viral polyprotein. Genetic or pharmacological inhibition of proline hydroxylation impairs nascent viral polyprotein folding and induces its aggregation and degradation. Notably, such interventions prevent viral polysome remodelling and lower virus production. Our findings delineate the modular nature of polysome specialization at the virus-host interface and establish a powerful strategy to identify targets for selective antiviral interventions.

Scottish soldiers from the Battle of Dunbar 1650: A prosopographical approach to a skeletal assemblage.

After the Battle Dunbar between English and Scottish forces in 1650, captured Scottish soldiers were imprisoned in Durham and many hundreds died there within a few weeks. The partial skeletal remains of 28 of these men were discovered in 2013. Building on previous osteological work, here we report wide-ranging scientific studies of the remains to address the following questions: Did they have comparable diet, health and disease throughout their lives? Did they have common histories of movement (or lack of movement) during their childhoods? Can we create a collective biography of these men? Strontium and oxygen isotope analysis of tooth enamel investigated childhood movement. Carbon and nitrogen isotope analysis of incrementally sampled dentine addressed childhood diet and nutrition. Metaproteomic analysis of dental calculus investigated oral microbiomes and food residues; this was complemented by microscopic analysis of debris in calculus from ingested materials. Selected individuals were examined for dental microwear. The extent of hydroxylation of proline in collagen was examined as a potential biomarker for scurvy. An osteobiography for each man was created using the full range of data generated about him, and these were synthesised using an approach based on the historical method for a collective biography or prosopography. The childhood residences of the men were primarily within the Midland Valley of Scotland, though some spent parts of their childhood outside the British Isles. This is concordant with the known recruitment areas of the Scottish army in 1650. Their diets included oats, brassicas and milk but little seafood, as expected for lowland rather than highland diets of the period. Childhood periods of starvation or illness were almost ubiquitous, but not simultaneous, suggesting regionally variable food shortages in the 1620s and 1630s. It is likely there was widespread low-level scurvy, ameliorating in later years of life, which suggests historically unrecorded shortages of fruit and vegetables in the early 1640s. Almost all men were exposed to burnt plant matter, probably as inhaled soot, and this may relate to the high proportion of them with of sinusitis. Interpersonal violence causing skeletal trauma was rare. Based on commonalities in their osteobiographies, we argue that these men were drawn from the same stratum of society. This study is perhaps the most extensive to date of individuals from 17th century Scotland. Combined with a precise historical context it allows the lives of these men to be investigated and compared to the historical record with unprecedented precision. It illustrates the power of archaeological science methods to confirm, challenge and complement historical evidence.

Proline hydroxylation in collagen supports integrin binding by two distinct mechanisms

Collagens are the most abundant extracellular matrix proteins in vertebrates and have a characteristic triple-helix structure. Hydroxylation of proline residues is critical for helix stability, and diminished prolyl hydroxylase activity causes wide-spread defects in connective tissues. Still, the role of proline hydroxylation in the binding of collagen receptors such as integrins is unclear. Here, we isolated skin collagen from genetically modified mice having reduced prolyl 4-hydroxylase activity. At room temperature, the reduced proline hydroxylation did not affect interactions with the recombinant integrin α2I domain, but at 37 °C, collagen hydroxylation correlated with the avidity of α2I domain binding. Of note, LC-MS/MS analysis of isolated skin collagens revealed no major changes in the hydroxyproline content of the main integrin-binding sites. Thus, the disrupted α2I domain binding at physiological temperatures was most likely due to structural destabilization of the collagenous helix. Integrin α2I binding to the triple-helical GFPGER motif was slightly weaker than to GFOGER (O = hydroxyproline). This phenomenon was more prominent when α1 integrin was tested. Integrin α1β1 expressed on CHO cells and recombinant α1I domain showed remarkably slower binding velocity and weaker avidity to GFPGER when compared with GFOGER. Structural modeling revealed the critical interaction between Arg-218 in α1I and the hydroxyproline residue in the integrin-binding motif. The role of Arg-218 was further validated by testing a variant R218D α1I domain in solid-phase binding assays. Thus, our results show that the lack of proline hydroxylation in collagen can affect integrin binding by a direct mechanism and via structural destabilization of the triple helix.

Glutaminolysis Promotes Collagen Translation and Stability via α-Ketoglutarate-mediated mTOR Activation and Proline Hydroxylation.

Glutaminolysis is the metabolic process of glutamine, aberration of which has been implicated in several pathogeneses. Although we and others recently found a diversity of metabolic dysregulation in organ fibrosis, it is unknown if glutaminolysis regulates the profibrotic activities of myofibroblasts, the primary effector in this pathology. In this study, we found that lung myofibroblasts demonstrated significantly augmented glutaminolysis that was mediated by elevated glutaminase 1 (Gls1). Inhibition of glutaminolysis by specific Gls1 inhibitors CB-839 and BPTES as well as Gls1 siRNA blunted the expression of collagens but not that of fibronectin, elastin, or myofibroblastic marker smooth muscle actin-α. We found that glutaminolysis enhanced collagen translation and stability, which were mediated by glutaminolysis-dependent mTOR complex 1 activation and collagen proline hydroxylation, respectively. Furthermore, we found that the amount of the glutaminolytic end product α-ketoglutarate (α-KG) was increased in myofibroblasts. Similar to glutaminolysis, α-KG activated mTOR complex 1 and promoted the expression of collagens but not of fibronectin, elastin, or smooth muscle actin-α. α-KG also remarkably inhibited collagen degradation in fibroblasts. Taken together, our studies identified a previously unrecognized mechanism by which a major metabolic program regulates the exuberant production of collagens in myofibroblasts and suggest that glutaminolysis is a novel therapeutic target for treating organ fibrosis, including idiopathic pulmonary fibrosis.

Nonantibiotic Effects of Fluoroquinolones in Mammalian Cells

Fluoroquinolones (FQ) are powerful broad-spectrum antibiotics whose side effects include renal damage and, strangely, tendinopathies. The pathological mechanisms underlying these toxicities are poorly understood. Here, we show that the FQ drugs norfloxacin, ciprofloxacin, and enrofloxacin are powerful iron chelators comparable with deferoxamine, a clinically useful iron-chelating agent. We show that iron chelation by FQ leads to epigenetic effects through inhibition of α-ketoglutarate-dependent dioxygenases that require iron as a co-factor. Three dioxygenases were examined in HEK293 cells treated with FQ. At sub-millimolar concentrations, these antibiotics inhibited jumonji domain histone demethylases, TET DNA demethylases, and collagen prolyl 4-hydroxylases, leading to accumulation of methylated histones and DNA and inhibition of proline hydroxylation in collagen, respectively. These effects may explain FQ-induced nephrotoxicity and tendinopathy. By the same reasoning, dioxygenase inhibition by FQ was predicted to stabilize transcription factor HIF-1α by inhibition of the oxygen-dependent hypoxia-inducible transcription factor prolyl hydroxylation. In dramatic contrast to this prediction, HIF-1α protein was eliminated by FQ treatment. We explored possible mechanisms for this unexpected effect and show that FQ inhibit HIF-1α mRNA translation. Thus, FQ antibiotics induce global epigenetic changes, inhibit collagen maturation, and block HIF-1α accumulation. We suggest that these mechanisms explain the classic renal toxicities and peculiar tendinopathies associated with FQ antibiotics.

Interaction of prolyl 4-hydroxylase with synthetic peptide substrates. A conformational model for collagen proline hydroxylation.

With the aim of understanding the structural basis for the substrate specificity of collagen prolyl 4-hydroxylase, we have studied the conformational features of synthetic oligopeptide substrates and their interaction with the enzyme purified from chicken embryo. Circular dichroism and infrared spectral data, taken in conjunction with relevant crystal structure data, indicated an equilibrium mixture of the polyproline-II (PP-II) helix, the beta-turn, and the random coil conformations in aqueous and trifluoroethanol solutions of the "collagen-related" peptides: t-Boc-Pro-Pro-Gly-Pro-OH, t-Boc-Pro-Pro-Gly-Pro-NHCH3, t-Pro-Pro-Gly-Pro-Pro-OH, t-Boc-Pro-Pro-Ala-Pro-OH, and t-Boc-Pro-Pro-Gln-Pro-OCH3, where t-Boc is tert-butoxycarbonyl. In another set of peptides related to elastin, t-Boc-Val-Pro-Gly-Val-OH and t-Boc-Gly-Val-Pro-Gly-Val-OH, the data indicated the beta-structure, rather than the PP-II helix, was in equilibrium with the beta-turn. Kinetic parameters for the enzymatic hydroxylation of the peptides showed that as a group, the first (proline-rich) set of peptides has higher Km values and lower Vmax and Kcat/Km values than the valine-rich peptides. Data on the inhibition of hydroxylation of the standard assay substrate (Pro-Pro-Gly)10 by the oligopeptides pointed to common binding sites for the peptides. Hydroxyproline-containing peptides had no effect on the hydroxylation of the standard substrate, showing the absence of product inhibition. Based on these and earlier data, we propose that in collagen and related peptides, a supersecondary structure consisting of the PP-II helix followed by the beta-turn is the minimal structural requirement for proline hydroxylation. The PP-II structure would aid effective interaction at the substrate binding subsites, while the beta-turn would be essential at the catalytic site of the enzyme. In elastin and related peptides, the beta-strand structure may be interchangeable with the PP-II structure. This conformational model for proline hydroxylation resolves the discrepancies in earlier proposals on the substrate specificity of prolyl 4-hydroxylase. It is also consistent with the available information on the active site geometry of the enzyme.

In vitro hydroxylation of proline in the collagen of the cysticercus of Taenia solium

1. 1. In vitro hydroxylation of proline in cysticercus collagen was carried out using two different vertebrate enzymes. 2. 2. Chick embryo enzyme is more active on cysticercus collagen than new-born rabbit enzyme. 3. 3. Hydroxylation of cysticercus collagen is more efficient at 40 than at 37°C. 4. 4. No prolyl hydroxylase activity was detected in cysticercus extracts. 5. 5. Collagen from the adult tapeworms Taenia solium and T. saginata lack hydroxyproline. 6. 6. SLS crystallites from T. solium and T. saginata collagen show the same band pattern as cysticercus collagen.

Coordinate regulation of collagen and proteoglycan synthesis in costal cartilage of scorbutic and acutely fasted, vitamin C-supplemented guinea pigs

The effects of ascorbic acid deficiency and acute fasting (with ascorbate supplementation) on the synthesis of collagen and proteoglycan in costal cartilages from young guinea pigs was determined by in vitro labeling of these components with radioactive proline and sulfate, respectively. Both parameters were coordinately decreased by the second week on a vitamin C-free diet, with a continued decline to 20–30% of control values by the fourth week. These effects were quite specific, since incorporation of proline into noncollagenous protein was reduced by only 30% after 4 weeks on the deficient diet. The time course of the decrease in collagen and proteoglycan synthesis paralleled the loss of body weight induced by ascorbate deficiency. Hydroxylation of proline in collagen synthesized by scorbutic costal cartilage was reduced to about 60% of normal relatively early, and remained at that level thereafter. Neither collagen nor proteoglycan synthesis was returned to normal by the addition of ascorbate (0.2 m m) to cartilage in vitro. Administration of a single dose of ascorbate to scorbutic guinea pigs increased liver ascorbate and restored proline hydroxylation to normal levels by 24 h, but failed to increase the synthesis of collagen or proteoglycan. Synthesis of both extracellular matrix components was restored to control levels after four daily doses of ascorbate. A 96-h total fast, with ascorbate supplementation, produced rates of weight loss and decreases in the synthesis of these two components similar to those produced by acute scurvy. There was a linear correlation between changes in collagen and proteoglycan synthesis and changes in body weight during acute fasting, scurvy, and its reversal. These results suggest that it is the fasting state induced by ascorbate deficiency, rather than a direct action of the vitamin in either of these two biosynthetic pathways, which is the primary regulatory factor.

Structural aspects of hydroxyproline-containing proteins.

The occurrence of hydroxyproline (Hyp) in collagen, C1q and acetylcholineesterase (AChE) raises important questions concerning the role of this unusual imino acid in the structure and function of these proteins. Available data on collagen indicate that Hyp is necessary for the normal secretion of the protein after its synthesis and for the integrity of the triple-helical conformation. Studies from our laboratory have dealt with the structural aspects of the posttranslational conversion of proline to hydroxyproline in collagen mediated by prolyl hydroxylase. We proposed that the beta-turn conformation at the Pro-Gly segments in the nascent procollagen molecule are the sites of the enzymatic hydroxylation and that this conformation changes over to the collagen-like helix as a result of the hydroxylation process. Recently, we have provided additional experimental support to our proposal by a) synthesizing specific beta-turn oligopeptides containing the Pro-Gly as well as Pro-Ala and Pro-DAla sequences and showing that these act as inhibitors of the enzymatic hydroxylation of a synthetic substrate and b) demonstrating, by circular dichroism spectroscopy, the occurrence of a conformational change leading to the triple-helix as a direct consequence of proline hydroxylation in a non-helical polypeptide substrate. We have also observed that the acquisition of hydroxylation results in a significant enhancement of the rate of folding of the polypeptide chain from the unfolded to the triple-helical conformation. We believe that our observations on proline hydroxylation in collagen should also be applicable to C1q and acetylcholineesterase both of which share the general structural and functional properties of collagen in their "tail" regions. Using the techniques employed in collagen studies, one should be able to assess the role of hydroxyproline in the folding, structural stabilities and functions of C1q and AChE. This would also involve the study of the unhydroxylated and hydroxylated precursors of these proteins which may share common structural features with their collagen counterparts. Finally, a systematic study of hydroxyproline-containing peptides and polypeptides has been initiated by us so as to understand the exact manner in which Hyp participates in the formation and stability of the triple-helical conformation in the proteins in which it occurs.

Effect of 2-aminoethyl-L-cysteine on collagen accumulation in isolated hepatic granulomas.

Hydroxylysine residues are important structural components of collagen as sites of glycosylation and cross-linking. Because hydroxylysine is formed from lysine by post-translational enzymatic hydroxylation, we examined the effects of incorporation of a lysine analogue that could interfere with hydroxylation. For this purpose, we synthesized 35S-labeled 2-aminoethyl cysteine. By addition of this compound to a smooth muscle cell culture, we obtained radiolabeled collagen which, upon acid hydrolysis, yielded the intact labeled analogue, thus demonstrating incorporation of the compound into collagen. Using a model hepatic collagen synthesizing system, freshly isolated granulomas derived from mouse livers after 8 weeks of infection with Schistosoma mansoni, incubation over a period of 7 days with [14C]proline to measure collagen production, demonstrated a reduction in acid-precipitable [14C]hydroxyproline per milligram DNA in granuloma cultures incubated with 2-aminoethyl cysteine compared to control cultures, and the effect was dose-related. That this decrease was selective for collagen accumulation over noncollagenous proteins was demonstrated by the finding of a significant reduction in the [14C]hydroxyproline/[14C] proline ratios compared to controls. A maximum incorporation of 10 residues of analogue/1000 residues was achieved. While the extent of hydroxylation of proline in collagen remained unchanged, hydroxylation of lysine decreased with increasing amounts of analogue incorporated. We conclude that in our system, 2-aminoethyl cysteine is incorporable into collagen. In a dose-related fashion, it selectively diminishes collagen accumulation compared to control.

Conformational implications of enzymatic proline hydroxylation in collagen.

In 1979 it was proposed that prolyl hydroxylase (prolyl-glycyl-peptide,2-oxoglutarate:oxygen oxidoreductase, EC 1.14.11.2) recognizes the beta-turn conformation in nascent procollagen chains and that the hydroxylation process involves a conformational change resulting in "straightening" of the beta-turn segments into the linear triple-helical conformation of native collagen. We present experimental data that verify both these postulates. The following peptides were synthesized and studied for their conformation and interaction with prolyl hydroxylase: tBoc-Pro-Gly-Ala-OH, tBoc-Pro-Gly-Val-OH, tBoc-Gly-Val-Pro-Gly-Val-OH, and tBoc-Pro-DAla-Ala-OH. Spectral data showed that these peptides preferred a beta-turn conformation. All of them acted as inhibitors of the enzyme; the pentapeptide also acted as a substrate. To mimic the biosynthetic event, a collagen model polypeptide, (Pro-Pro-Gly)10, was incubated at 37 degrees C with purified prolyl hydroxylase and the necessary cosubstrates and cofactors at pH 7.8. A progressive change from the initially nonhelical to the triple-helical conformation, as monitored by CD spectra and gel filtration, occurred during the course of proline hydroxylation. In addition to leading to increased thermal stability of the triple-helical conformation in (Pro-Pro-Gly)10 and (Pro-Pro-Gly)5, the enzymatic incorporation of the hydroxyproline residues was found to enable these polypeptides to fold into this conformation faster than the unhydroxylated counterparts. These conformational implications of proline hydroxylation in collagen may also be of use in the study of the complement subcomponent Clq and of acetylcholine esterase which contain collagen-like regions in them.

Normal hydroxylation of proline in collagen synthesized by skin fibroblasts from a patient with prolidase deficiency.

The extent of hydroxylation of proline in collagen synthesized and secreted into the culture medium by skin fibroblasts derived from a patient with prolidase deficiency has been examined and found to be normal. It would seem likely that to a considerable extent the urinary proline-containing dipeptides apparent in this condition are derived from sources other than collagen.

A new method for determining the extent of proline hydroxylation by measuring changes in the ratio of [4- 3H]:[ 14C]proline in collagenase digests

A new method has been developed to determine the extent of proline hydroxylation in collagen. This method relies on the measurement of changes in the ratio of [ 3H]:[ 14C]-proline in bacterial collagenase digests, resulting from specific loss of tritium from the 4- trans position of the proline during hydroxylation. Proline hydroxylation can be measured in the presence of large amounts of noncollagen proteins, and simultaneous quantitation of the rates of collagen and noncollagen protein synthesis is obtained. The dual-labeled proline method is simple, rapid, and accurate. There was a very good correlation between this method and both ion-exchange chromatography and chloramine-T oxidation/toluene extraction procedures currently used for the measurement of proline hydroxylation.

Control of collagen production by human diploid lung fibroblasts.

The fibroblast is a differentiated mesenchymal cell which produces and exports collagen, a macromolecule that plays a critical structural role in the function of most organs. To evaluate the control soft tissue fibroblasts have over collagen production, HFL-1, a diploid human lung cell strain, was studied during periods of rapid cell growth and relatively slow growth over 25 population doublings. To minimize environmental influences, the extracellular milieu of the cells was kept constant throughout the study period. Rates of collagen production per cell per unit time were quantitated by labeling HFL-1 with [14C]proline and measuring the production of [14C]hydroxyproline after taking into consideration the specific activity of [14C]proline within the free intracelllular proline pool and the per cent hydroxylation of proline residues in newly synthesized collagen. Although the specific activity of intracellular free proline and the per cent hydroxylation of proline in collagen varied considerably depending on the growth rates of the cells, collagen production by HFL-1 was constant, even during periods of rapid cell growth. Thus, under conditions of a stable environment, populations of soft tissue fibroblasts rigidly control their collagen production. In cultures that maintained a constant doubling time, this stability was maintained over at least 25 population doublings, suggesting that on the average, collagen production appears to be tightly controlled and dissociated from the events and sequelae of cell division.

Effects of glucocorticosteroids on cultured human skin fibroblasts—IV: Specific decrease in the synthesis of collagen but no effect on its hydroxylation

Confluent cultures of normal baby foreskin fibroblasts were exposed for 6 days to hydrocortisone-17-butyrate (5 μg/ml)or to clobetasol-17-propionate (1 or μg/ml). On day 5 [ 3H]proline was added to the cultures and on day 6 both medium and cell layer were analyzed for [ 3H]protein and protein-bound [ 3H]hydroxyproline. The synthesis of labeled protein was little affected, while that of labeled collagenous protein was greatly depressed, as compared to that of the control cells. This depression occurred for the collagenous protein present in the cell layer as well as for that released into the growth medium. This effect was not accompanied by a decrease in cellular prolyl hydroxylase activity or in collagen proline hydroxylation.

Constancy of proline hydroxylation in collagen synthesized at different temperatures by poikilotherms

Previous experiments have suggested that the body temperature of an organism may in part regulate the hydroxyproline content of its collagen. In order to test this hypothesis, the hydroxyproline content of the collagen synthesized at different temperatures by fish cells in culture and by intact amphibian larvae was measured. High levels of prolyl hydroxylation were found at both low and high temperatures, which eliminates the possibility that temperature can directly modulate prolyl hydroxylation in vivo. Synthesis and hydroxylation of collagen appear to be coordinated so that unstable underhydroxylated molecules are not produced under normal circumstances.

Dependence of the differentiated state on the cellular environment: modulation of collagen synthesis in tendon cells.

In an adequate environment, primary avian tendon cells are capable of retaining both the full expression of differentiated function and a correct morphological orientation for 1 week in culture. At high density and in the presence of ascorbate, they are fully stabilized in that they devote 25-30% of their total protein synthesis to collagen, a level comparable to that in tendon cells in ovo. However, either at low density or in medium without ascorbate, they synthesize collagen at only a third of this level. If plated on a collagen matrix, these cells will orient themselves in a manner similar to that of tendon cells in vivo. Furthermore, they are capable of fully modulating the percentage of collagen synthesis upon addition or removal of ascorbate and serum. The variation in the percentage of collagen produced is a result of alterations in collagen synthesis rather than of changes in total protein synthesis or hydroxylation of proline in collagen. Primary avian tendon cells, therefore, provide a suitable model for understanding the stability of the differentiated state, the mechanism of action of ascorbate, and the regulation of collagen biosynthesis.

Proline and hydroxyproline excretion and vitamin C status in elderly human subjects.

1. Plasma and buffy-coat vitamin C, urinary proline, hydroxyproline, creatinine and total amino acid concentrations were meausred in 23 healthy elderly subjects at intervals of 3 months. 2. There was a strong positive correlation between plasma vitamin C and buffy-coat vititamin C. 3. There were not significant correlations between plasma or buffy-coat vitamin CPAMIN C. 3. There were not significant correlations between plasma or buffy-coat vitamin C and total urinary hydroxyproline, whether expressed on a creatinine basis or on a total amino acid basis. Similarly, no significant correlations could be detected involving the proline/hydroxyproline ratio in urine hydrolysates. 4. There was a significant negative correlation between plasma or buffy-coat vitamin C and total urinary proline, when expressed per unit of total urinary proline, when expressed per unit of total urinary proline, when expressed per unit of total amino acids in the hydrolysates. This correlation was not observed with unhydrolysed urine, and it appeared to reside in the diffusible fraction, part of whose proline could be liberated by prolidase digestion. In addition, in the man, there was some evidence for a positive correlation between plasma or buffy-coat vitamin C and the ratio of total urinary amino acids to creatinine. 5. These results support the view that poor vitamin C status in elderly humans may be associated with a defect in collagen proline hydroxylation, reflected by increased excretion of proline-rich, collagen-derived peptides. If this interpretation is correct, it indicates a potential defect in connective tissue repair, and could form the basis of a functional test for subclinical vitamin C deficiency.