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Background 1,5-Diamino-2-hydroxy-pentane (2-OH-PDA), as a new type of aliphatic amino alcohol, has potential applications in the pharmaceutical, chemical, and materials industries. Currently, 2-OH-PDA production has only been realized via pure enzyme catalysis from lysine hydroxylation and decarboxylation, which faces great challenges for scale-up production. However, the use of a cell factory is very promising for the production of 2-OH-PDA for industrial applications, but the substrate transport rate, appropriate catalytic environment (pH, temperature, ions) and separation method restrict its efficient synthesis. Here, a strategy was developed to produce 2-OH-PDA via an efficient, green and sustainable biosynthetic method on an industrial scale. Results In this study, an approach was created for efficient 2-OH-PDA production from l-lysine using engineered E. coli BL21 (DE3) cell catalysis by a two-stage hydroxylation and decarboxylation process. In the hydroxylation stage, strain B14 coexpressing l-lysine 3-hydroxylase K3H and the lysine transporter CadB-argT enhanced the biosynthesis of (2S,3S)-3-hydroxylysine (hydroxylysine) compared with strain B1 overexpressing K3H. The titre of hydroxylysine synthesized by B14 was 2.1 times higher than that synthesized by B1. Then, in the decarboxylation stage, CadA showed the highest hydroxylysine activity among the four decarboxylases investigated. Based on the results from three feeding strategies, l-lysine was employed to produce 110.5 g/L hydroxylysine, which was subsequently decarboxylated to generate a 2-OH-PDA titre of 80.5 g/L with 62.6% molar yield in a 5-L fermenter. In addition, 2-OH-PDA with 95.6% purity was obtained by solid-phase extraction. Thus, the proposed two-stage whole-cell biocatalysis approach is a green and effective method for producing 2-OH-PDA on an industrial scale. Conclusions The whole-cell catalytic system showed a sufficiently high capability to convert lysine into 2-OH-PDA. Furthermore, the high titre of 2-OH-PDA is conducive to separation and possesses the prospect of industrial scale production by whole-cell catalysis.

Stromal stiffening accompanies malignancy, compromises treatment and promotes tumour aggression. Clarifying the molecular nature and the factors that regulate stromal stiffening in tumours should identify biomarkers to stratify patients for therapy and interventions to improve outcome. We profiled lysyl hydroxylase-mediated and lysyl oxidase-mediated collagen crosslinks and quantified the greatest abundance of total and complex collagen crosslinks in aggressive human breast cancer subtypes with the stiffest stroma. These tissues harbour the highest number of tumour-associated macrophages, whose therapeutic ablation in experimental models reduced metastasis, and decreased collagen crosslinks and stromal stiffening. Epithelial-targeted expression of the crosslinking enzyme, lysyl oxidase, had no impact on collagen crosslinking in PyMT mammary tumours, whereas stromal cell targeting did. Stromal cells in microdissected human tumours expressed the highest level of collagen crosslinking enzymes. Immunohistochemical analysis of biopsies from a cohort of patients with breast cancer revealed that stromal expression of lysyl hydroxylase 2, an enzyme that induces hydroxylysine aldehyde-derived collagen crosslinks and stromal stiffening, correlated significantly with disease specific mortality. The findings link tissue inflammation, stromal cell-mediated collagen crosslinking and stiffening to tumour aggression and identify lysyl hydroxylase 2 as a stromal biomarker.

The diagenesis of a bone in the postmortem period causes an identifiable deterioration in histology. This degradation is characterized by a collagenous alteration, which can be observed very early. In order to develop a method for determining a postmortem interval for medico-legal use, two ribs collected from six human bodies were studied prospectively over 2 years. Each bone was studied after staining with Sirius red to demonstrate the degradation of collagen as a function of time. This study demonstrated a time-based bone alteration characterized by the architectural degradation of the lamellar bone, without any microbial influence in this postmortem period. The staining was carried out by using Sirius red and correlated this alteration with a collagenic degradation by chemical hydrolysis owing to the affinity of this dye to the amino acids lysine, hydroxylysine, and arginine. Our work asserts that human bone samples that were studied in a controlled environment and analyzed for 24 months underwent a diagenetic trajectory whose main element was collagen hydrolysis.

The suitability of HPLC with fluorescent derivatization for simultaneous determination of eight collagen-related bone turnover biomarkers, 4-hydroxy-l-proline (OHP), pyridinoline (PYD), hydroxylysine (Hyl), deoxypyridinoline, and their glycosides, galactosyl-Pyd (Gal-Pyd, GP), galactosyl-glucosyl-Pyd, β-1-galactosyl-O-hydroxylysine (Gal-Hyl, GH), and α-1,2-glucosyl-galactosyl-O-hydroxylysine (GGH), in diverse clinical samples was investigated. 5-Carboxyfluorescein succinimidyl ester (CFSE) was chosen as the fluorescent labeling reagent. Various parameters affecting derivatization and separation were optimized. Under optimal conditions, maximum derivatization could be achieved in 25 min at 30 °C, and complete baseline separation could be completed within 25 min, with relative standard deviation values for corrected peak areas less than 3.5 % for intraday assay and 4.2 % for interday assay, respectively. The limits of detection were determined to be 0.1–1.7 nM, which are well below the expected concentrations in the real samples. The method has been used for the quantitative determination of eight target analytes in various clinical samples, including cell cultures of normal human osteoclast, preosteoblast, osteoclastoma cells and serum and urine fluids from patients with fracture, osteitis fibrosa, or osteoclastoma as well as healthy subjects, with spiked recoveries of 96–103 %. This original application of HPLC–fluorescence detection represents a powerful tool for investigating the dynamics of metabolic imbalance of these biomarkers for bone turnover under physiological and pathophysiological conditions.

Collagen was extracted from several tissues (muscle, skin, bone, alimentary tract, gill, fin, hepatopancreas, and air bladder) of the tiger pufferfish Takifugu rubripes, and the content and solubility of the collagen extracted from each tissue were examined. Collagen content in ordinary muscle was 0.95 ± 0.07 % of wet tissue, which is lower than that reported for other fish species even though tiger pufferfish meat has a tough texture. The solubility of collagen extracted from the muscle and skin was relatively high, and collagen accounted for 47.2 ± 7.8 and 70.8 ± 8.1 % of wet tissue, respectively. In contrast, the solubility of the collagen extracted from bone was the lowest of all the tissues examined, being only 5.7 ± 0.8 % of total wet tissue. The extent of hydroxylation of proline and lysine residues was also examined. In most tissues, the extent of hydroxylation of the lysine residue in insoluble collagen was higher than that of acid-soluble collagen, indicating that hydroxylysine contributes to the stability of collagen. This is the first report of collagen contents, solubility, and extent of hydroxylation of proline and lysine residues in collagen extracted from different tissues of one organism. It is possible that hydroxylysine-derived collagen cross-links play a critical role in the stability of collagen in dilute acetic acid.

Collagen IV comprises the predominant protein network of basement membranes, a specialized extracellular matrix, which underlie epithelia and endothelia. These networks assemble through oligomerization and covalent crosslinking to endow mechanical strength and shape cell behavior through interactions with cell-surface receptors. A recently discovered sulfilimine (S=N) bond between a methionine sulfur and hydroxylysine nitrogen reinforces the collagen IV network. We demonstrate that peroxidasin, an enzyme found in basement membranes, catalyzes formation of the sulfilimine bond. Drosophila peroxidasin mutants have disorganized collagen IV networks and torn visceral muscle basement membranes, pointing to a critical role for the enzyme in tissue biogenesis. Peroxidasin generates hypohalous acids as reaction intermediates, suggesting a paradoxically anabolic role for these usually destructive oxidants. This work highlights sulfilimine bond formation as what is to our knowledge the first known physiologic function for peroxidasin, a role for hypohalous oxidants in tissue biogenesis, and a possible role for peroxidasin in inflammatory diseases.

Lysyl oxidase is the extracellular enzyme that catalyzes oxidative deamination of peptidyl-lysine residues in elastin precursors, and lysine and hydroxylysine residues in collagen precursors to form peptidyl-aldehydes. These aldehydes then spontaneously condense to crosslink collagen and elastin and thereby allow the formation of a mature and functional extracellular matrix. In the present study, cryosections made from aseptic immune-induced periapical lesions experimentally generated in laboratory rats were examined by immunohistochemistry to investigate whether lysyl oxidase protein expression is altered in inflamed oral tissues. Periapical lesions are experimentally induced endodontic lesions of tooth roots. In addition, the effect of administration of a mixture of fibroblast growth factor (FGF)-2 and insulin-like growth factor (IGF)-1 into these lesions on lysyl oxidase expression was determined. Lysyl oxidase expression was found to be increased in non-mineralized connective tissue adjacent to inflamed lesions. Morphometric analyses indicated that maximum lysyl oxidase expression occurred at a discrete distance from the lesion not exceeding 350 microm from the inflammatory cells. Staining was associated with mesenchymal cells with a fibroblastic morphology. No lysyl oxidase staining was found near teeth where no lesion was induced. Application of a mixture of FGF-2 and IGF-1 resulted in a further twofold increase in lysyl oxidase expression. These results provide a new in vivo model to study lysyl oxidase regulation, and suggest that inflammatory cells may control lysyl oxidase expression in oral tissues, possibly by a mechanism involving secretion of cytokines and other factors, probably contributing to the regulation of extracellular matrix accumulation.

The ovariectomized rat is the most commonly used animal model of human postmenopausal osteoporosis, exhibiting a high rate of bone turnover with resorption exceeding formation. At present, bone turnover is quantified directly by dynamic histomorphometry. The aim of the present study was to determine whether the measurement of the urinary output of some specific bone collagen catabolites--pyridinolines and hydroxylysine glycosides--could be used to indirectly monitor the initial phase of bone turnover increase in ovariectomized 90-day-old rats. Ninety-day-old female rats were randomly divided into three groups (n = 6): ovariectomized, sham-operated and non-treated controls. Urine samples (24 h) were collected 6 days before surgery and twice weekly for the 4 weeks following ovariectomy. Urinary excretion of pyridinoline (PYD), deoxypyridinoline (DPD), glucosyl-galactosyl-hydroxylysine (GGHYL) and galactosyl-hydroxylysine (GHYL) were measured. As expected, ovariectomy was associated with a significant decrease in bone mineral density in both the proximal tibial and distal femoral metaphysis. Compared with both sham-operated and control animals, ovariectomized rats showed significant increases in PYD, GGHYL, and GHYL urinary output 8 days after surgery and in DPD output after 15 days. These changes were maintained throughout the study. The results confirm that measurement of the urinary excretion of pyridinolines and hydroxylysine glycosides represents a powerful tool for detecting the onset of bone turnover in ovariectomized 90-day-old rats.

The concentration dependence of the influx ofl-lysine in excised roots ofArabidopsis thaliana seedlings was analyzed for the wild-type (WT) and two mutants,rlt11 andraec1, which had been selected as resistant to lysine plus threonine, and to S-2-aminoethyl-l-cysteine, respectively. In the WT three components were resolved: (i) a high-affinity, low-capacity component [K m = 2.2 μM;V max = 23 nmol·(g FW)−1·h−1]; (ii) a low-affinity, high-capacity component [K m = 159 μM;V max = 742 nmol·(g FW)−1·h−1]; (iii) a component which is proportional to the external concentration, with a constant of proportionalityk = 104 nmol·(g FW)−1 h−1];·mM−1. The influx ofl-lysine in the mutants was lower than in the WT, notably in the concentration range 0.1–0.4 mM, where it was only 7% of that in the WT. In both mutants the reduced influx could be fully attributed to the absence of the low-affinity (high-K m ) component. This component most likely represents the activity of a specific basic-amino-acid transporter, since it was inhibited by several other basic amino acids (arginine, ornithine, hydroxylysine, aminoethylcysteine) but not byl-valine. The high-affinity uptake ofl-lysine may be due to the activity of at least two general amino acid transporters, as it was inhibitable byl-valine, and could be further dissected into two components with a high affinity (K i = 1–5 μM; and a low affinity (K i = 0.5–1mM) forl-valine, respectively. Therlt11 andraecl mutant have the same phenotype and the corresponding loci were mapped on chromosome 1, but it is not yet clear whether they are allelic.

Aim of this study was to investigate whether osteoclast activity changes as a consequence of even mild physiological perturbation of plasma calcium as such induced by an oral calcium load. Osteoclast activity was determined indirectly by measuring, in spot urines at two and four hours after oral calcium load, the urinary excretion of hydroxylysylpyridinoline (Pyr), deoxylysylpyridinoline (D-Pyr), hydroxyproline (Hyp) and galactosyl-hydroxylysine (GHyl). The occurrence of the metabolic perturbation of plasma calcium homeostasis was assessed by measuring three indexes: i.e. calcemic response, PTH reduction and calciuric response at times following oral calcium loading. A significant fall of urinary D-Pyr and Pyr followed the perturbation of calcium homeostasis induced by the oral calcium load in two groups of healthy young adult and postmenopausal women. The highest mean percent reduction was observed for D-Pyr and was quantitatively similar in the two groups. Since urinary D-Pyr is the most specific bone resorption marker, it may be inferred that the perturbation of plasma calcium homeostasis induced by an oral calcium load is able to acutely inhibit osteoclast activity. This supports the view that osteoclasts are involved in the short-term error correction of plasma calcium.

Aim of the study was to compare urinary galactosyl-hydroxylysine (GHyl), deoxypyridinoline (D-Pyr) and pyridinoline (Pyr) before and after 5 to 9 months of hormone replacement therapy (HRT) in postmenopausal women. The urinary markers were measured by HPLC in the second void of fasting samples and were expressed as ratio to creatinine. GHyl was also expressed as a ratio to glucosylgalactolysyl-hydroxylysine (GGHyl). After short-term hormone replacement therapy, urinary D-Pyr fell significantly, but Pyr and GHyl, also when expressed as a ratio to GGHyl, remained unmodified. We conclude that GHyl and Pyr are not useful markers in monitoring the bone response to HRT in postmenopausal women.

The composition of collagen was analyzed and the degree of lysyl hydroxylation of individual collagen chains was determined in four osteosarcomas and two osteofibrous dysplasias. In addition, the tumor proliferation (number of mitoses, proliferating-nuclear-antigen-positive cells, MIB) as well as the response to chemotherapy (morphological regression grade) were checked. All tumors contained a high proportion of collagen III and, in all but one osteosarcoma, pepsin-extracted collagens I and III were overmodified. Furthermore, the proportion of diglycosides in collagen I was about four times higher than in controls. The collagen composition and modification resembled those of bones at early stages of human development. One osteosarcoma and both osteofibrous dysplasias were in the normal range of lysyl hydroxylation. There was no correlation between the collagen properties and the histopathological marker of tumor proliferation.

Sixty men with spinal cord injury who had developed pressure ulcers in the past but whose skin was intact when they joined the study were followed for 2 years, or until a pressure ulcer developed. Forty of the men were contacted every 4-6 weeks to answer questions about their skin care practices and to provide a 24 hour urine sample. The others were only contacted at the beginning and the end of the study to answer a questionnaire and to provide a urine sample. Changes in skin collagen metabolism were monitored by measuring urinary excretion of a metabolite, glucosyl-galactosyl hydroxylysine (glu-gal Hyl), corrected for creatinine excretion. Sustained increases in levels of glu-gal Hyl excretion were detected at least 2 months and as much as 5 months in advance of overt clinical signs of ulcer development. Increased excretion of glu-gal Hyl was significantly associated (p < 0.05) with the development of a pressure ulcer. An increase in the urinary excretion of glu-gal Hyl is an indication of increased degradation of skin collagen. Body mass index (weight/height2) of 33% of subjects with pressure ulcers, and 12% of those without, was at least one standard deviation below the mean of all subjects. Thirty-six percent of those who smoked developed ulcers, while only 26% of the nonsmokers did.(ABSTRACT TRUNCATED AT 250 WORDS)

The C1 complex of human complement comprises two loosely interacting subunits, C1q and the Ca(2+)-dependent C1s-C1r-C1r-C1s tetramer. With a view to gain information on the nature of the ionic interactions involved in C1 assembly, we have studied the effects of the chemical modifications of charged residues of C1q or the tetramer on their ability to reconstitute the C1 complex. Treatment of C1q with pyridoxal-5'-phosphate, acetic anhydride, and citraconic anhydride, as well as with cyclohexanedione and diethylpyrocarbonate, inhibited its ability to associate with C1s-C1r-C1r-C1s. Treatment of the collagen-like fragments of C1q with the same reagents yielded the same effects. Treatment of C1s-C1r-C1r-C1s with 1-ethyl-3-[-3-(dimethylamino) propyl] carbodiimide also prevented C1 assembly, through modification of acidic amino acids which were shown to be located in C1r. Further studies on the location of the interaction sites within C1q, using ligand-blotting and competition experiments with synthetic peptides, were unsuccessful, suggesting that these sites are contributed to by two or three of the C1q chains. It is concluded that C1 assembly involves interactions between acidic amino acids of C1r and lysine (hydroxylysine) and arginine residues located within the collagen-like region of C1q. Sequence comparison with mannan binding protein, another collagen-like molecule which binds the C1s-C1r-C1r-C1s tetramer, suggests Arg A38, and HyL B32, B65, and C29 of C1q as possible interaction sites.

The collagen study includes the analysis of its characteristic amino acids: proline, hydroxyproline, lysine, hydroxylysine. HPLC offers an interesting device if associated with on-line radiometric detection for the determination of radiolabelled amino acids in the case of metabolism studies. To avoid pre or post-column derivatization which may be poorly quantitative in the case of the hydrolysate of unpurified samples, we developed an ion-paired reversed-phase chromatography using a C8 column (econosphere C8 5µm, length: 250 mm, ID: 4.6 mm from Alltech Ass.) and an elution carried out with an acetonitrile gradient in heptane-sulfonate solution. A direct detection at 210 nm was used. Nineteen amino acids were separated within 40 min. Lag time was 7.3 min between hydroxyproline and proline, and 6.9 min between hydroxylysine and lysine. In the case of radiolabelled amino acid, there was a linear correlation (r = 0.92) between HPLC and ion-exchange chromatography.

Pepsin-solubilized collagen I from skin and bone was analyzed with regard to its thermal stability as a triple helical molecule in solution and after in vitro fibril formation. Collagen I from human control bone was compared with samples showing deficiencies or surplus in the degree of hydroxylation of lysine. The helix to coil transitions were studied by circular-dichroism measurements and limited trypsin digestion. Melting of fibrils from standardized in vitro self-assembly was investigated turbidimetrically. Human control bone collagen I has a maximum transition rate (Tm) at 43.3 degrees C in 0.05% acetic acid. This is 1.9 degrees C above control skin (Tm = 41.4 degrees C), most likely, due to a higher degree of prolyl hydroxylation--0.48 in bone vs. 0.41 in skin collagen I. Lysyl overhydroxylation of human and mouse bone collagen I appears to reduce the Tm slightly (approximately 1 degree C). Underhydroxylated bone collagen has a Tm which is 2 degrees C below control. Melting temperatures of in vitro formed fibrils are an indication for higher thermostability in parallel with an increase of lysyl hydroxylation. Accordingly, the melting temperature of such fibrils from human control skin, 49.3 degrees C, exceeds control bone by 1.4 degrees C. The degree of lysyl hydroxylation in these samples is 0.14 and 0.10, respectively. Further underhydroxylation (0.06) reduced it down to 45.4 degrees C, while extensive overhydroxylation did not continue to increase the thermal stability of fibrils.

A method is described for measurement of urinary hydroxylysine glycosides (galactosyl-hydroxylysine, or GHYL, and glucosyl-galactosyl-hydroxylysine, or GGHYL) by reversed phase chromatography after purification of the sample by solid phase extraction. Urinary levels of hydroxylysine glycosides has been correlated with collagen catabolism, and the urinary GGHYL: GHYL ratio is indicative of the metabolism of connective tissue. The GGHYL:GHYL ratio might, therefore, be a promising indicator of bone mass loss. The method described is analytically reliable and enables the measurement of a possible marker for diagnosis and follow-up of osteoporosis.

The excretion of collagen metabolites and circulating androgens was measured in ten males suffering from progressive systemic sclerosis. Significantly higher levels of sex hormone-binding globulin, total testosterone and dihydrotestosterone (DHT) (P less than 0.01), and total oestradiol (P less than 0.05), were found in patients when compared with age-matched controls. Urinary excretion of hydroxyproline in patients was found to correlate significantly with total testosterone (P = 0.035), with DHT (P = 0.005) and dehydroepiandrosterone-sulphate (DHEAS) (P = 0.034). Similarly the hydroxyproline peptide fraction was found to correlate significantly with total testosterone (P = 0.037), with DHT (P = 0.005) and with DHEAS (P = 0.008). Hydroxylysine peptide in the urine correlated significantly with free testosterone (P = 0.035) and DHT (P = 0.040). Oestrogens did not correlate with urinary excretion of collagen metabolites. These findings suggest that androgens may play a role in the pathogenesis of scleroderma in male patients.

Metabolic profiling by capillary liquid chromatography-electrospray mass spectrometry was used to monitor shifts in the proferrioxamine profiles of Erwinia amylovora in response to externally supplied potential proferrioxamine precursors, selected stable-isotope-labeled precursors and atypical precursors. Based on the qualitative and quantitative shifts in the proferrioxamine profiles, lysine and arginine are unambiguous, and agmatine, ornithine, diaminobutyric acid and the corresponding C3-5 diamines are highly likely precursors for proferrioxamine biosynthesis in E. amylovora. 5-Hydroxylysine (Hyl), a recently discovered growth inhibitor for E. amylovora, suppresses proferrioxamine production. The Hyl-induced growth inhibition can be reversed by basic amino acids. The basic amino acids also partly restore proferrioxamine synthesis.

In practical pediatric endocrinology assessment of current height velocity (HV) plays an important role for diagnosis and treatment of growth disorders. However, to date, there is no quick and simple method to determine HV. Urinary excretion of Galactosyl-hydroxylysine (GHLy), a marker of osteoclast activity, has been shown to closely reflect the activity of bone metabolism in adults. In this study we tested the hypothesis that GHLy can also be used to evaluate skeletal growth in children. GHLy was measured in 24h-urines of 74 healthy children, 21 girls with Turner's syndrome (TU) and 15 children with growth hormone deficiency (GHD). HV was calculated from two measurements of body height 6 to 12 months apart. When related to body weight, the 24h excretion of GHLy showed a highly significant correlation to HV in normal children (r=0.75, p 0.05). Data for weight-adjusted daily excretion of GHLy were transformed to standard deviation scores (SDS) to allow for comparison in different age groups. The mean SDS of both GHD and TU were significantly below zero (−1.31 and −1.83 respectively; p

Collagen from the calvarium of a 2-week-old infant with osteogenesis imperfecta and dentine collagen from a first permanent molar of an 11-year-old boy with dentinogenesis imperfecta were prepared by methods which had previously been used for collagens from normal hard tissues. Amino-acid analysis, carried out on hydrolysates of these preparations, showed the same amino-acids as are found in normal collagens. There was no gross deficiency in any particular amino acid and the compositional differences accompanying the pathological conditions appeared to be small. The collagen from osteogenesis imperfecta had a 7–9% deficiency in the abundant amino acids and an excess of amino acids with lipophilic side chains, compared with collagen from normal bone, which suggested that up to 10% of other protein material could be present in osteogenesis imperfecta. In dentinogenesis imperfecta there was a similar but smaller decrease in the amounts of hydroxyproline and alanine but there was an increase in the amounts of glycine and proline present. An excess of hydroxylysine was found in both diseased states compared with the corresponding normal hard tissue collagens. A 12% deficiency in weight recovery compared with nitrogen recovery may be explained by carbohydrate material responsible for argyrophilia.

Biopsies of a cutaneous osteoma and of normal-looking skin from a 1-year-old girl were studied for histological appearance and collagen biochemistry. The mineralized tissue contained a matrix similar to bone: Only type I collagen, with a hydroxylysine content (0.48%) higher than in the skin (0.35%) and dihydroxylysinonorleucine as the major reducible crosslink. As expected, the normal skin adjacent to the lesions contained type I and type III collagen and as major crosslinks hydroxylysinonorleucine and histidinohydroxymerodesmosine. Histological studies showed the presence of woven bone with very little trabeculation. Numerous active osteoblasts were laying down a rapidly calcified non-lamellar matrix. Osteocytes and multinucleated osteoclasts were also noted. The study demonstrates the osseous nature of the lesion and suggests that an abnormal cell differentiation is associated with this form of osteoma.

A glycoprotein of an apparent molecular weight of 250,000 has been purified from human amniotic fluid. Its amino acid composition includes trace amounts of hydroxyproline but no hydroxylysine. It has relatively high contents of aspartic acid, glutamic acid, glycine and leucine. It contains approximately 6% hexose, 2% sialic acid, and 4% glucosamine. The hexoses are galactose, mannose and fucose. No glucose and galactosamine can be detected. This high-molecular-weight glycoprotein cross-reacts with an antiserum prepared in rabbits against a glycoprotein of similar size and composition isolated from the alveolar secretions of humans suffering from pulmonary alveolar proteinosis [20]. It therefore appears that the glycoprotein isolated from human amniotic fluid is similar in size, composition and immunological properties to the glycoprotein present in human alveolar secretions.

Hyphal walls were isolated from the Oomycete, Apodachlya sp. Microscope examination of wall preparations showed that they were clean and relatively free of cellulin granules. The principal wall constituents, accounting for more than half of wall weight, were beta-glucans with 1,3- and 1,6-glucosidic linkages. Apparently chitin was the second most abundant wall constituent (18%) and cellulose accounted for less than 10% of wall weight. Protein was a significant wall constituent (6.4%), and protein hydrolysis demonstrated nearly all common amino acids plus hydroxyproline; additionally, the unusual amino acid, hydroxylysine was tentatively identified. The lipid and ash constituents were small (1.7and 0.4%, respectively) and no particular significance was assigned to them. The possible occurrence of wall glycoproteins and the relationship between wall chemistry and systematics in Apodachlya and related genera were discussed.

Free amino acid chromatographic fingerprints obtained in automated analysers in 24-hour urines may help to quantitate muscle and collagen changes occurring after cerival spinal cord injury. The technique was used to compare the urine of six healthy and ten tetraplegic men, 18 to 25 years old, three of whom became paralysed in diving, one in a mountain fall, one one in the fall of a pipe, four in car accidents, and one in a football game. Nine weeks to 14 years after injury, the tetraplegics excreted less total free amino acids, lysine, and 3-methylhistidine than the controls, whereas hydroxyproline and hydroxylysine were similar in both groups. Values of urine 3-methylhistidine, measured 9, 13, 19 and 20 weeks post onset in one patient, and 9, 12, 16, 18, 23, and 26 weeks post onset in another, were related to the duration of paralysis. These results indicate that collagen and muscle metabolism may not be quantitated by measuring urine free amino acids.

This study was performed to compare the extractability of dwarf growth plate collagen and hexosamine and that of homozygous nonaffected Malamutes and to measure the activity of three of the enzymes involved in the post-translational modifications of the collagen molecule. No significant differences were found in the activity of prolyl hydroxylase or lysyl oxidase in the dwarf growth plates. Lysyl hydroxylase activity in the dwarf was decreased to 22% and 33% that of the activity present in the homozygous nonaffected growth plates. Amino acid analysis of the collagen isolated from dwarf growth plates failed to reveal any decrease in hydroxylysine content. Growth plates were extracted with either 1 M sodium chloride or 4 M guanidine hydrochloride. The extracts were applied to a DEAE-cellulose column. Amino acid analyses of the material which did not bind to DEAE revealed a slight decrease in the amount of guanidine-extractable hydroxyproline in the dwarf but a 60-fold increase in the amount of salt-extractable hydroxyproline in the dwarf growth plates. Material which eluted with 1 M sodium choloride was analyzed for hexosamine. There was a 10-fold increase in the amount of salt-extractable hexosamine present in the dwarf growth plates, whereas no significant differences were observed in the guanidine-extracted material. Hexosamine analysis of the growth plates revealed a significant increase in the total amount of hexosamine present in the dwarf growth plates. SDS-polyacrylamide gels of the material which did not bind to DEAE as well as the pepsin digested, 0.9M sodium chloride precipitated collagen demonstrated the presence of only type II collagen.

The urinary excretion of glucosyl-galactosyl-hydroxylysine and of galactosyl-hydroxylysine were studied in Paget's disease of bone and in primary hyperparathyroidism. Both metabolites were increased in these diseases. Although glucosyl-galactosyl-hydroxylysine is not abundant in bone collagen, it is possible that a part of it originates from calcified tissue.

The effects of streptozotocin diabetes on the collagen metabolism in rat glomerular basement membrane (GBM) have been investigated. The metabolic activity was measured in vivo by following the incorporation of 3H-proline into hydroxyproline over the course of 3 months. Under diabetic conditions, smaller amounts of labeled hydroxyproline were found in the GBM indicating that collagen synthesis is injured. The retarded disappearance of the tracer from the GBM is related to an additional alteration of the degradation resulting in a slower turnover. If collagen formation is reduced in streptozotocin diabetes, the thickening of the diabetic basement membranes can only explained by this disturbed catabolism. The amino acid composition of GBM was also altered after streptozotocin application. The diabetic animals showed an increase of hydroxylysine in this structure. The diabetic effects on the GBM presented were interpreted as a kind of accelerated aging of collagen.

A new glycoprotein with an apparent molecular weight of 130,000 was isolated, purified, and partially characterized from the pulmonary secretions which accumulate so massively in the lungs of patients suffering from pulmonary alveolar proteinosis. The amino acid analysis of the glycoprotein showed the presence of relatively high amounts of glycine, glutamic acid, aspartic acid, leucine, and valine, and small amounts of hydroxyproline, but no hydroxylysine. It contains approximately 6% hexose, 3% sialic acid, and 4% glucosamine. The neutral sugars are galactose, mannose, and fucose. This alveolar glycoprotein cross-reacted with an antiserum prepared in rabbits against a larger glycoprotein (250,000 mol wt) isolated from the same source, suggesting that the larger alveolar glycoprotein may be the precursor of the smaller one.

The insoluble collagen from methylcholanthrene induced sarcoma was isolated and characterized. It contains more glycine, hydroxyproline and acidic amino acids than normal connective tissue collagen. An anionic character of tumour collagen was stated (pI 6.1). No typical collagen subunits in this protein were found. The tumour collagen is strongly bound to acidic glycoprotein containing a significant amount of hydroxylysine. Such an insoluble complex is resistant to the dispersing action of EDTA. It dissociates during heating in concentrated urea.

A glycoprotein of an apparent molecular weight of 250,000 has been purified from the cell-free culture medium of human fetal lung Type II pneumocytes. Its amino acid composition shows the presence of trace amounts of hydroxyproline but no hydroxylysine. It is relatively rich in aspartic acid, glutamic acid, glycine and leucine. It contains about 6.0% hexose, 2.7% sialic acid and 3.7% glucosamine. The hexoses are galactose, mannose and fucose. No glucose or galactosamine can be detected. This high-molecular-weight glycoprotein cross-reacts with an antiserum prepared in rabbits against a glycoprotein of similar size and composition isolated from the alveolar secretions of patients suffering from pulmonary alveolar proteinosis [20]. It appears that the high-molecular-weight alveolar glycoprotein is a secretory product of the alveolar Type II cells.

Amino acid analyses of carefully dissected samples of the articular cartilage of postnatal chickens indicates the presence of significant amounts of a collagen with a low hydroxylysine content. It was found that 9 M LiCl solutions preferentially extract the components of a collagen whose components when identified by ion exchange chromatography and amino-acid analyses, established that they were derived from Type I collagen. Type II collagen was essentially insoluble in solutions of 9 M LiCl, but could be solubilized in 4 M CaCl2 after prior treatment of the cartilage with pepsin. The αl (II) components were then isolated and characterized by their behavior during ion exchange chromatography and by their amino acid composition. The findings establish that the articular cartilage of postnatal chickens contains substantial amounts of Type I collagen and suggest that the nature and stability of the crosslinkages in Type I collagen of cartilage differs significantly from those of Type II collagen of cartilage. Moreover, it is also postulated that the nature of the major intermolecular cross-linkages in the Type I collagen of cartilage are similar to those present in Type I collagen of bone rather than those present in the Type I collabens of skin and other connective tissues.

Methods are described for the isolation of galactosyl hydroxylysine and glucosylgalactosyl hydroxylysine from collagen and for the quantitative analysis of these hydroxylysine glycosides. The isolation procedure, based upon gel filtration and preparative ionpaired reversed-phase high-performance liquid chromatography, is simple and rapid in comparison with existing methods, allowing the production of tens of milligrams of each glycoside in a single day. Quantitative analysis of the hydroxylysine glycosides is effected by reversed-phase high-performance liquid chromatography with precolumn derivatization (usingo-phthal-dialdehyde) and fluorescence detection.

The biosynthess of collagen and elastin by chick embryo skin of 11–16 days of development was studied. An increased uptake of (14C) proline and (14C) lysine on day 12 was observed in collagen peptides and in elastin extracted from the skin samples. (14C)hydroxyproline followed the same pattern as the uptake of its precursor into the collagenase-digested peptide fraction. The conversion of (14C)lysine into (14C)hydroxylysine was low. The conversion of (14C)glutamic acid and (14C)glutamine into (14C)proline and (14C)hydroxyproline by the skin of chicken embryos may be an important pathway. As far as (14C)proline and (14C)lysine is concerned elastin showed a similar pattern of changes during the maturation process.

The fluorescence curves of partial acid hydrolysates of dentin and skin collagens were compared with the curves of aqueous solutions of 21 amino acids, and 16 small peptides which might be obtained from the backbone chain of collagen. The collagen degradation mixtures showed maximum emission at 410 mμ. Of the amino acids, only hydroxylysine had an emission peak at 410 mμ. Glycyl peptides in which glycine was amino-terminal also gave maximum emission in this region. Such peptides, present in high concentration in collagen hydrolysates, may account for much of the observed fluorescence.

Oxidative degradation of decalcified bovine dentin matrix with alkaline sodium metaperiodate solubilizes a substantial part of the matrix. The soluble portion includes a fraction containing more than 75% of the covalently-bound phosphorus present in the native dentin. Preparative scale electrophoresis of the solubilized dentin on polyacrylamide gels led to the isolation of a group of four strongly anionic components (I–IV). Analyses showed each component to be a protein characterized by a high content of phosphate groups, serine and aspartic acid, and, with one exception (II), small amounts of proline and hydroxyproline. Each component contained one residue of hydroxylysine per mole. In addition to the periodate resistant hydroxylysine, III and IV also contained periodate resistant glucose and galactose. On the basis of the presence of hydroxyproline in varying amount, and hydroxylysine on a mole per mole basis, it is suggested that components I, III and IV consist of collagen backbone fragments linked to a phosphoprotein moiety with a high content of serine and aspartic acid. The periodate-resistant hexoses and the hydroxylysine probably represent the linkage point. Component II is a phosphoprotein moiety essentially free of a collagen fragment. These data provide direct evidence that dentin collagen contains a covalently bound non-collagen polypeptide attachment of highly acidic character.

The susceptibility of Bacillus subtilis to amino acid analogues was found to be markedly influenced by the carbon source used in the test media. Thialysine inhibited the bacterium with a greater number of carbon sources than the other two analogues tested. 5-Hydroxylysine was inhibitory with glycerol, lactose, D-xylose, L-arabinose and soluble starch while ethionine showed toxicity with lactose, D-xylose and L-arabinose. None of these analogues were toxic at the levels tested when D-galactose was used as carbon source. The bacterium was not susceptible to thialysine with glycerol, to 5-hydroxylysine with L-arabinose and to ethionine with lactose.

The biosynthesis of trans-3- and trans-4-hydroxyprolines and 5-hydroxylysine in animal cells requires polypeptide proline or lysine, enzymes and cofactors including iron, and possibly involves peroxidatic intermediates1. Several laboratories have reported the presence of low-molecular-weight hydroxyproline and hydroxylysine peptides in cell and organ cultures2–5. We found that these small peptides contained the trans-3 and cis-4 isomers of hydroxyproline as well as trans-4 ones and that their production was not completely inhibited by α, α-dipyridyl, an iron chelator and effective inhibitor of enzyme-mediated hydroxylations5. It is known that oxygen or hydrogen peroxide in the presence of metal can hydroxylate proline and other aromatic compounds6–11. We show here that reduced oxygen derivatives can hydroxylate both free and poly peptide-bound proline and lysine, and that scavengers of hydroxyl radicals suppress, but do not completely inhibit, this reaction. Reduced oxygen derivatives can be generated in normal and pathological circumstances12, and some of the low-molecular-weight hydroxyproline and hydroxylysine peptides found in cell and organ cultures might be derived from these derivatives and therefore do not reflect collagen turnover, but rather some other cellular activity.

THE serum protein C1q is a subcomponent of the first component of the complement system. Combination of C1q with antigen–antibody complexes activates the other subcomponents of C1 (C1r and C1s) and sets in motion a series of events which results in activation of the entire complement system. The primary structure of C1q is unusual in that 40% of its amino acid sequence is composed of repeating X-Y-Gly triplets where X is often proline and Y often hydroxyproline or hydroxylysine, indicating a striking similarity to collagen1,2. It has also been proposed that these collagen polypeptide-like stretches in C1q associate in triple helical fashion3. Because of the similarities between these portions of C1q and collagen the present investigation was undertaken to determine whether C1q is synthesised by collagen-producing cells, namely fibroblasts.

CONTROVERSIAL results1–5 on the hydroxylysine content of wool were due to the difficulty of analysis of hydroxylysine at very low concentrations. We have now been able to identify and estimate hydroxylysine in wool by application of new methods.

ALL collagens from vertebrates and invertebrates, except the annelid and nematode cuticles, contain significant amounts of hydroxylysine1. Examination of a large number of these hydroxylysine-containing proteins has so far revealed that all contain carbohydrate units, consisting of either galactose or glucosylgalactose (2-O-α-D-glucopyranosylgalactose) linked by β-glycosidic bonds to a variable number of the hydroxylysine residues2,3.

Nicolet and Shinn1 and Van Slyke et al.2 have studied the action of periodic acid on hydroxyaminoacids, and in the cases of serine and hydroxylysine have demonstrated the rapid formation of one molecule of formaldehyde. Block and Boiling3 have made use of the rapid formation of one molecule of acetaldehyde from threonine on oxidation with lead tetracetate for determining this amino-acid in protein hydrolysates.

THE synthesis of collagen involves several enzymatic modifications of the polypeptide precursor of collagen, protocollagen1–4. First, certain proline and lysine residues in protocollagen are hydroxylated in similar enzymatic reactions involving molecular 02, Fe2+, α-ketoglutarate and ascorbic acid5–8. Much of the hydroxylation occurs after completed polypeptide chains have been released from the polyribosomal complexes9–11. Some hydroxylysine residues are then glycosylated by the stepwise addition of glucose and galactose, to form disaccharide appendages on the polypeptide chain12 and this glycosylation may be essential for the secretion of collagen into the extracellular matrix13,14. Although there are conflicting reports concerning the regulatory role of the hydroxylating enzymes15–20, no information is available on the genetic controls of collagen biosynthesis.

THE periodic acid Schiff technique has been used histochemically primarily as a means of localizing the 1,2 glycol linkage in tissue sections1. It is known, however, that periodic acid is also capable of cleaving the 1,2 hydroxylamino linkage with the production of some aldehyde radicals, and although the exact reaction mechanism is not understood, it is believed by some to react in a somewhat similar manner with a number of amino-acids possessing structurally neither of these linkages. The amino-acids serine and threonine, as well as the “hydroxylysine” of Van Slyke, fall into the first category, whereas cystine, methionine, tryptophane, glycine, alanine, tyrosine, histadine, aspartic and glutamic acids fall into the latter group2. This investigation was initiated to study specifically this phase of the periodic acid Schiff mechanism, and although the results were not conclusive, it was felt that a brief description of the experiments might be of interest to others working in this particular field.

Five disorders of collagen have been characterized. In four of these disorders the patients have had symptoms of Ehlers Danlos. Three families with lysyl hydroxylase deficiency have previously been reported. We are reporting an additional case diagnosed in infancy. This infant was investigated as a floppy baby but nerve and muscle were normal. Lysyl hydroxylase activity measured in sonicates of dermal fibroblasts was 10% of normal. The deficiency of hydroxylysine results in abnormal cross-linking of collagen molecules which may account for the symptomatology. The patient we are reporting eventually developed symptoms of Ehlers Danlos. This type of Ehlers Danlos is an autosomal recessive disorder in which one might expect to make an intrauterine diagnosis. Collagen abnormalities may thus be a cause of floppy baby syndrome and the delineation of a specific defect should be the goal of diagnostic studies.

Collagen metabolism was studied in 9 children with hereditary nephritis and 10 family members with microscopic hematuria. The diagnosis was confirmed in all 9 children by percutaneous renal biopsy. No previous studies exist defining the collagen turnover via the utilization of the urinary OHLG's, hydroxylysylgalactosyl-glucose (HGG) and hydroxylysyl-galactose (HG), in hereditary nephritis. Mean urinary OHLG's values in the biopsy group was 113 uM/g creatinine as compared to 15 control children of 74 uM/g creatinine. The mean urinary values of 128 u M/g creatinine for the siblings were comparable to the nephritis group. Two additional patients with the Nail-Patella Syndrome also demonstrated increased urinary excretion of OHLG's. The significantly increased urinary excretion of these metabolites, HGG and HG, support the thesis of an altered collagen disorder. This study suggests that urinary hydroxylysine glycosides may be used as a useful marker in the recognition of the early stage of hereditary nephritis.

IN order to estimate the lysine content of wool, using partition chromatography on silica gel1, a sample of ethanol- and ether-extracted wool (Lincoln Hogg, tips removed) was hydrolysed at the boil with 5·l7 N hydrochloric acid for twenty-four hours. An aliquot was then treated with 1-fluoro-2: 4-dinitrobenzene and an unsuccessful attempt made to separate the di-2: 4 dinitrophenyllysine from the other amino-acid derivatives by means of suitable solvents2, owing to considerable overlapping of the various 2: 4-dinitrophenyl amino-acids. However, making use of Porter and Sanger's copper complex technique for the preparation of e N-2: 4-dinitrophenyllysine3, a relatively simple method for the quantitative estimation of lysine was developed, using a hydrolysate solution containing the equivalent of 5–10 mgm. of the original wool.