New marker of bone resorption: hydroxyproline-containing peptide High-performance liquid chromatographic assay without hydrolysis as an alternative to hydroxyproline determination: a preliminary report

A high-performance liquid chromatographic (HPLC) assay for a urinary hydroxyproline-containing peptide (hydroxyproline peptide, HypP) is described. This peptide represents about 50% of urinary hydroxyproline-containing peptides. Its concentration and total 4-hydroxyproline (Hyp) concentration evaluated in 325 urine samples have been shown to be closely correlated (r = 0.972; y = 0.499x − 1.5), which may indicate that the two markers provide the same information. The HypP assay, similar to Hyp assay, is carried out without hydrolysis of urine samples. After the blocking of primary amino acids by o-phthaldialdehyde (OPA) and derivatization of secondary amino acids by 9-fluorenylmethyl chloroformate (FMOC-Cl), the FMOC derivatives of HypP and 3,4-dehydroproline (internal standard) were separated on a strong anion-exchange column and detected fluorimetrically. HypP concentration was calculated by measurement of peak-area ratios of HypP and the hydroxyproline standard. The HypP/creatinine (mmol/mol) ratio in fasting urine samples from healthy adults was found to be 8.2 (S.D. = 1.6, n = 33) in 27–44-year-old premenopausal women and 6.9 (S.D. = 1.7, n = 21) in 28–49-year-old men.     

Effect of silver nanoparticles and hydroxyproline,administered in ovo,on the development of blood vessels and cartilage collagen structure in chicken embryos

It has been considered that concentrations of certain amino acids in the egg are not sufficient to fully support embryonic development of modern broilers. In this study we evaluated embryo growth and development with particular emphasis on one of the major components of connective tissue, collagen. Experiments were performed on Ross 308 chicken embryos from 160 fertilised eggs. Experimental solutions of silver nanoparticles (Ag), hydroxyproline solution (Hyp) and a complex of silver nanoparticles with hydroxyproline (AgHyp) were injected into albumen, and embryos were incubated until day 20. An assessment of the mass of embryo and selected organs was carried out followed by measurements of the expression of the key signalling factors’ fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor-A (VEGF-A). Finally, an evaluation of collagen microstructure using scanning electron microscopy was performed. Our results clearly indicate that Hyp, Ag and AgHyp administered in ovo to chicken embryos did not harm embryos. Comparing to the control group, Hyp, Ag and the AgHyp complex significantly upregulated expression of the FGF-2 at the mRNA and protein levels. Moreover, Hyp, Ag and, in particular, the complex of AgHyp significantly increased blood vessel size, cartilage collagen fibre lattice size and bundle thickness. The general conclusion from this study is that AgHyp treatment may help to build a stronger and longer lasting form of collagen fibres.     

Non-proline-accumulating rice mutants resistant to hydroxy-L-proline

Summary Three rice (Oryza sativa L.) mutants resistant to hydroxy-L-proline (Hyp), HYP 101, HYP 202 and HYP 203, were selected from an ethylene imine mutagenized M₂ population of the original variety, Nipponbare, and their biochemical and genetical characteristics were investigated. The sensitivity of the mutants to Hyp could be clearly differentiated from that of the original variety when seeds were germinated and cultured with 10^–410^–3 M Hyp for 10 days. A difference in Hyp sensitivity was also observed among the HYP mutant lines, HYP 101 being the most resistant line. When free amino acids in seeds and 15-day-old seedlings were analyzed, the composition of the amino acids in the mutants was somewhat different from that found in the original variety. However, free proline accumulation was not detected in either the HYP mutants or the original variety. In each mutant line, HYP resistance was transmitted with a single recessive nuclear gene (hpr). These results suggest that the mechanism of Hyp resistance controlled by the recessive gene do not involve free proline accumulation.Abbreviations Hyp hydroxy-L-proline - T-Pro thioproline     

Highly sensitive determination of N-terminal prolyl dipeptides, proline and hydroxyproline in urine by high-performance liquid chromatography using a new fluorescent labelling reagent, 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride

A highly sensitive pre-column HPLC method for simultaneous determination of prolyl dipeptides, Pro and Hyp in urine was developed. The analytes were labelled with 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride at 70°C for 20 min. The derivatives separated on tandem reversed-phase columns by a gradient elution and were monitored with fluorescence detection at 318 nm (excitation) and 392 nm (emission). The detection limits for prolyl dipeptides, Pro and Hyp were 1–5 fmol/injection (S/N=3). Urine samples were treated with o-phthalaldehyde, followed by purification on a Bond Elut C₁₈ column before conducting the labelling reaction. Pro–Hyp, Pro–Gly and Pro–Pro were identified as prolyl dipeptides in urine. The within-day and between-day relative standard deviations were 1.5–4.8 and 1.7–5.8%, respectively. The concentrations of Pro–Hyp, Pro–Gly, Pro–Pro, Pro and Hyp in normal human urine were 97.6±28.2, 2.74±1.48, 2.08±1.13, 6.71±3.34 and 2.30±1.59 nmol/mg creatinine, respectively.     

Effect of Alfalfa Wilts on the Hydroxyproline Content in Cell Wall

The Hyp content was studied in cell wall of alfalfa susceptible and resistant strains on the 3rd, the 7th and on the 14th day after inoculation with Verticillium albo-atrum or Corynebacterium michiganense pv. insidiosum. The changes of Hyp content after inoculation with both pathogens were markedly expressed in alfalfa roots. Resistant plants of R 337 strain responded to inoculation with V. albo-atrum or C. michiganense pv. insidiosum by the decrease of Hyp content mainly on the 3rd and on the 7th day. On the 14th day after inoculation Hyp content practically did not differ from that of the control. Susceptible plants of S 354 and S 321 srains responded to inoculation with wilt pathogens by the slight decrease of Hyp content at the 3rd day after inoculation. A significant increase of Hyp content was found on the 7th and mainly on the 14th day after inoculation in comparison with control plants. The cell wall Hyp content was also determined with 7 R-strains and 7 S-strains at 120 days after inoculation with both pathogens. In each R and S strain two categories of plants were used for chemical analyses: Wilt-free plants (0 to 1 classes) and diseased, wilted plants (2 to 6 classes). In the resistant alfalfa strains no differences in Hyp content between the wilt-free and diseased plants were found. In the susceptible alfalfa strains the Hyp content was significantly higher in roots of diseased plants comparing with the wilt-free ones. Only negligible changes in Hyp content were registered in the overground parts of all inoculated alfalfa strains.     

Hypericin‐PDT‐induced rapid necrotic death in human squamous cell carcinoma cultures after multiple treatment

PDT (photodynamic therapy) has been used for the treatment of NMCC (non‐melanoma cutaneous cancer) particularly, human SCC (squamous cell carcinoma). However, the nature of the photosensitizer, the activation light source and the mode of cell death induced post‐PDT remains elusive. We tried to optimize PDT using the light‐activated (320–400 nm) St John's Wort‐derived compound, Hyp (hypericin). The work highlights the potential mode of cell death and the increased efficacy of the technique associated with multiple Hyp‐PDT treatment. SCC cells were exposed to different concentrations of Hyp and activated with light at 1 J/cm² for 1 or 2 days. Thereafter with the optimum dose of Hyp proliferation, ROS (reactive oxygen species), and apoptosis were analysed by XTT [2,3‐bis‐(2‐methoxy‐4‐nitro‐5‐sulfophenyl)‐2H‐tetrazolium‐5‐carboxanilide] assay, FACS analysis and Fluorescent/Phase contrast microscopy was carried out for morphological studies. Hyp‐PDT produces more ROS after 1 day compared with 2 days and the mode of cell death is a necrotic caspase‐independent mechanism. We propose a novel ‘double‐hit/2‐day’ strategy to reduce the viability in SCC using Hyp‐based PDT as an adjunctive treatment modality.     

Engineering ‘designer’ glycomodules for boosting recombinant protein secretion in tobacco hairy root culture and studying hydroxyproline‐O‐glycosylation process in plants

The key technical bottleneck for exploiting plant hairy root cultures as a robust bioproduction platform for therapeutic proteins has been low protein productivity, particularly low secreted protein yields. To address this, we engineered novel hydroxyproline (Hyp)‐O‐glycosylated peptides (HypGPs) into tobacco hairy roots to boost the extracellular secretion of fused proteins and to elucidate Hyp‐O‐glycosylation process of plant cell wall Hyp‐rich glycoproteins. HypGPs representing two major types of cell wall glycoproteins were examined: an extensin module consisting of 18 tandem repeats of ‘Ser‐Hyp‐Hyp‐Hyp‐Hyp’ motif or (SP4)₁₈ and an arabinogalactan protein module consisting of 32 tandem repeats of ‘Ser‐Hyp’ motif or (SP)₃₂. Each module was expressed in tobacco hairy roots as a fusion to the enhanced green fluorescence protein (EGFP). Hairy root cultures engineered with a HypGP module secreted up to 56‐fold greater levels of EGFP, compared with an EGFP control lacking any HypGP module, supporting the function of HypGP modules as a molecular carrier in promoting efficient transport of fused proteins into the culture media. The engineered (SP4)₁₈ and (SP)₃₂ modules underwent Hyp‐O‐glycosylation with arabino‐oligosaccharides and arabinogalactan polysaccharides, respectively, which were essential in facilitating secretion of the fused EGFP protein. Distinct non‐Hyp‐O‐glycosylated (SP4)₁₈‐EGFP and (SP)₃₂‐EGFP intermediates were consistently accumulated within the root tissues, indicating a rate‐limiting trafficking and/or glycosylation of the engineered HypGP modules. An updated model depicting the intracellular trafficking, Hyp‐O‐glycosylation and extracellular secretion of extensin‐styled (SP4)₁₈ module and AGP‐styled (SP)₃₂ module is proposed.     

Amino acid concentrations in hypothalamic and caudate nuclei during microwave-induced thermal stress: Analysis by microdialysis

Exposure to radiofrequency radiation (RFR) may produce thermal responses. Extracellular amino acid concentrations in the hypothalamus (Hyp) and caudate nucleus (CN) were measured by using in vivo microdialysis before and during exposure to RFR. Under urethane anesthetic, each rat was implanted stereotaxically with a nonmetallic microdialysis probe and temperature probe guides and then placed in the exposure chamber. The rat laid on its right side with its head and neck placed directly under the wave guide. Temperature probes were placed in the left brain, right brain, face (subcutaneously), left tympanum, and rectum. Each microdialysis sample was collected over a 20 min period. The microdialysis probe was perfused for 2 h before the rat was exposed to 5.02 GHz radiation (10 μs pulse width, 1000 pulses/s). The right and left sides of the brain were maintained at approximately 41.2 and 41.7 °C, respectively, throughout a 40 min exposure period. Initially when the brain was being heated to these temperatures, the time-averaged specific absorption rates (SARs) for the right and left sides of the brain were 29 and 40 W/kg, respectively. Concentrations of aspartic acid, glutamic acid, serine, glutamine, and glycine in dialysate were determined by using high-pressure liquid chromatography with electrochemical detection. In the Hyp and CN, the concentrations of aspartic acid, serine, and glycine increased significantly during RFR exposure (P < .05). These results indicate that RFR-induced thermal stress produces a general change in the amino acid concentrations that is not restricted to thermoregulatory centers. Changes in the concentrations of glutamic acid (Hyp, P = .16; CN, P = .34) and glutamine (Hyp, P = .13; CN, P = .10) were not statistically significant. Altered amino acid concentrations may reveal which brain regions are susceptible to damage in response to RFR-induced thermal stress. Bioelectromagnetics 18:277–283, 1997. © 1997 Wiley-Liss, Inc.     

Reconstruction of tricarboxylic acid cycle in Corynebacterium glutamicum with a genome-scale metabolic network model for trans-4-hydroxyproline production

trans-4-Hydroxy- l -proline (Hyp) is an abundant component of mammalian collagen and functions as a chiral synthon for the syntheses of anti-inflammatory drugs in the pharmaceutical industry. Proline 4-hydroxylase (P4H) can catalyze the conversion of l -proline to Hyp; however, it is still challenging for the fermentative production of Hyp from glucose using P4H due to the low yield and productivity. Here, we report the metabolic engineering of Corynebacterium glutamicum for the fermentative production of Hyp by reconstructing tricarboxylic acid (TCA) cycle together with heterologously expressing the p4h gene from Dactylosporangium sp. strain RH1. In silico model-based simulation showed that α-ketoglutarate was redirected from the TCA cycle toward Hyp synthetic pathway driven by P4H when the carbon flux from succinyl-CoA to succinate descended to zero. The interruption of the TCA cycle by the deletion of sucCD-encoding the succinyl-CoA synthetase (SUCOAS) led to a 60% increase in Hyp production and had no obvious impact on the growth rate. Fine-tuning of plasmid-borne ProB* and P4H abundances led to a significant increase in the yield of Hyp on glucose. The final engineered Hyp-7 strain produced up to 21.72 g/L Hyp with a yield of 0.27 mol/mol (Hyp/glucose) and a volumetric productivity of 0.36 g·L ⁻¹·hr ⁻¹ in the shake flask fermentation. To our knowledge, this is the highest yield and productivity achieved by microbial fermentation in a glucose-minimal medium for Hyp production. This strategy provides new insights into engineering C. glutamicum by flux coupling for the fermentative production of Hyp and related products.     

Quantification of hydroxyproline in small amounts of skin tissue using isocratic high performance liquid chromatography with NBD-F as fluorogenic reagent

In order to determine the collagen content of small amounts of skin tissue, we developed a new, simple and highly sensitive method of measuring the quantity of hydroxyproline (Hyp) using isocratic high performance liquid chromatography (HPLC) with a fluorogenic agent, 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F). The recovery rate of Hyp and reproducibility of the assay were high, and the test was sensitive enough to detect Hyp in less than 1 mg of skin tissue. This method is clinically useful for ensuring accurate diagnosis and for monitoring specific skin conditions using small human skin samples collected in biopsies.     

Effects of hypericin on the structure and aggregation properties of β-amyloid peptides

We have determined the secondary structure of 1–40 β-amyloid peptides by Fourier-transform infrared spectroscopy (FTIR) and characterized the peptide photophysical properties before and after self-assembly by using intrinsic tyrosine steady-state and time-resolved fluorescence. All measurements were performed in the presence and absence of hypericin (Hyp), an exogenous natural polycyclic pigment that has been shown to inhibit fibril formation and has also been used as a fluorescent probe. We monitored the time course of the aggregation process measuring 405 nm light diffusion at 90° and used thioflavin T to reveal the presence of fibrils. FTIR quantitative analysis evidenced a prevalent random conformation at t = 0 with and without Hyp. Fibrils showed a predominant parallel β-sheet structure and a small percentage of α-helix. The results of fluorescence measurements showed that Hyp does significantly interact with peptides in β-sheet conformation. In conclusion, hypericin does hinder the formation of fibrils, but the percentages of parallel β-sheets were not significantly different from those found in samples not treated with Hyp.     

Fluorometric determination of N-terminal prolyl dipeptides, proline and hydroxyproline in human serum by pre-column high-performance liquid chromatography using 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride

A highly sensitive HPLC method for the determination of prolyl dipeptides, Pro and Hyp in serum was developed. After deproteinization of serum and pretreatment with o-phthalaldehyde, the analytes were derivatized with 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride at 70°C for 10 min. The fluorescent derivatives of prolyl dipeptides, Pro and Hyp, were separated on tandem reversed-phase columns by a gradient elution at 55°C and detected by fluorescence measured at 318 nm (excitation) and 392 nm (emission). The detection limits for prolyl dipeptides were 2–5 fmol/injection (S/N=3). Pro–Hyp, Pro–Gly and Pro–Pro were identified as serum prolyl dipeptides. The within-day and between-day relative standard deviations were 1.5–7.9 and 2.4–10.8%, respectively. The recoveries were in the range of 90.8–97.3%. The concentrations of Pro–Hyp, Pro–Gly, Pro–Pro, Pro and Hyp in normal human serum (n=10) were 0.64±0.35, 0.078±0.047, 0.022±0.016, 177.0±43.0 and 11.1±3.5 μM, respectively. The concentrations of Pro–Hyp and Pro–Pro in serum of a patient with bone metastases of prostatic cancer were about three times and 50 times, respectively, higher than those in normal human serum.     

Heterotrimeric collagen peptides containing functional epitopes. Synthesis of single‐stranded collagen type I peptides related to the collagenase cleavage site

Synthetic collagen peptides containing larger numbers of Gly‐Pro‐Hyp repeats are difficult to purify by standard chromatographic procedures. Therefore, efficient strategies are required for the synthesis of higher molecular weight collagen‐type peptides. Applying the Fmoc/tBu chemistry, a comparative analysis of the standard stepwise chain elongation procedure on solid support with the procedure based on the use of the synthons Fmoc‐Gly‐Pro‐Hyp(tBu)‐OH and Fmoc‐Pro‐Hyp‐Gly‐OH was performed. The crude products resulting from the stepwise elongation procedure and from the use of Fmoc‐Gly‐Pro‐Hyp(tBu)‐OH clearly revealed large amounts of microheterogeneities that result from incomplete imino acid acylation as well as from diketopiperazine formation with cleavage of Gly‐Pro units from the growing peptide chain. Conversely, by the use of the Fmoc‐Pro‐Hyp‐Gly‐OH synthon, the quality of the crude products was significantly improved; moreover, protection of the Hyp side chain hydroxyl function is not required using the Fmoc/tBu strategy. With this optimized synthetic procedure, relatively large collagen‐type peptides were obtained in satisfactory yields as highly homogeneous compounds. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Effect of mercuric chloride on various hydroxyproline fractions in rat serum

Mercuric chloride (HgCl₂) disturbs the collagen metabolism in the body which is reflected by altered hydroxyproline fractions in the serum. The aim of the present investigation was to study the effect of HgCl₂ treatment on various hydroxyproline (Hyp) fractions in rat serum and the effect of 2,3-dimercapto-1-propane sulfonic acid (DMPS) treatment on serum Hyp fractions in HgCl₂ treated rats. Other parameters studied included body weight, food intake, water intake and kidney weight. Doses of HgCl₂ used were 0.1, 0.5, 1.0, 2.0, 3.0 mg/kg body weight and that of DMPS was 100 mg DMPS/kg body weight. All the doses of HgCl₂ used caused significant (p < 0.01) alterations in free, peptide-bound and protein-bound Hyp in the serum when compared with control rats but a dose of 2 mg/kg body weight caused significant (p < 0.001) alteration even in the total serum Hyp when compared to control rats. Administration of DMPS prior HgCl₂ treatment of rats sacrificed 24 h after the treatment caused a significant decrease of 52% (p < 0.01) in free Hyp when compared to similar HgCl₂ treated rats. DMPS treatment with HgCl₂ also caused an increase of 61% (p < 0.001) and 114% (p < 0. 001) in peptide- and protein-bound Hyp respectively, when compared to HgCl₂ treated rats sacrificed 24 h after mercuric chloride and DMPS treatment. Administration of DMPS followed by HgCl₂ to rats which were sacrificed 48 h later caused no significant change in the total and free Hyp when compared to HgCl₂ treated rats which were sacrificed 48 h after the treatment. But there was a significant decrease of 40% (p < 0.001) in peptide-bound Hyp and an increase in of 77% (p < 0.001) in protein-bound Hyp when compared to HgCl₂ treated rats sacrificed 48 h after the treatment. The present study shows that HgCl₂ treatment caused significant alterations in serum Hyp fractions reflecting disturbed composition of connective tissues which were not reversed by DMPS treatment. (Mol Cell Biochem 271: 159–165, 2005)     

Chassis engineering of Escherichia coli for trans-4-hydroxy-l-proline production

Microbial production of trans-4-hydroxy-l -proline (Hyp) offers significant advantages over conventional chemical extraction. However, it is still challenging for industrial production of Hyp due to its low production efficiency. Here, chassis engineering was used for tailoring Escherichia coli cellular metabolism to enhance enzymatic production of Hyp. Specifically, four proline 4-hydroxylases (P4H) were selected to convert l -proline to Hyp, and the recombinant strain overexpressing DsP4H produced 32.5 g l⁻¹ Hyp with α-ketoglutarate addition. To produce Hyp without α-ketoglutarate addition, α-ketoglutarate supply was enhanced by rewiring the TCA cycle and l -proline degradation pathway, and oxygen transfer was improved by fine-tuning heterologous haemoglobin expression. In a 5-l fermenter, the engineered strain E. coliΔsucCDΔputA-VHb_(L)-DsP4H showed a significant increase in Hyp titre, conversion rate and productivity up to 49.8 g l⁻¹, 87.4% and 1.38 g l⁻¹ h⁻¹ respectively. This strategy described here provides an efficient method for production of Hyp, and it has a great potential in industrial application.     

Analysis of hydroxyproline isomers and hydroxylysine by reversed-phase HPLC and mass spectrometry

Collagens, the most abundant mammalian proteins, contain a high content of hydroxylated amino acids, such as, 3- and 4-cis-/trans-hydroxyproline (Hyp) and 5-hydroxylysine (Hyl). Whereas the global content of 4-Hyp was studied by amino acid analysis, no technique to determine all five hydroxyamino acids simultaneously in collagens has been reported. Here, we report the separation of all five hydroxyamino acids as well as two Hyp epimers from all other proteinogenic amino acids after derivatization with N(2)-(5-fluoro-2,4-dinitrophenyl)-l-valine amide (l-FDVA) by RPC-UV-ESI-MS. The general applicability of this method is shown for three Hyp-containing peptides as well as collagen type I.     

Effective inactivation of food pathogens Listeria monocytogenes and Salmonella enterica by combined treatment of hypericin-based photosensitization and high power pulsed light

Aims: The aim of this study was to evaluate the inactivation efficiency of Listeria monocytogenes ATC_L3C 7644 and Salmonella enterica serovar Typhimurium strain DS88 by combined treatment of hypericin (Hyp)‐based photosensitization and high power pulsed light (HPPL). Methods and Results: Cells were incubated with Hyp (1 × 10^?5 or 1 × 10^?7 mol l^?1) in PBS and illuminated with a light λ = 585 nm. For the combined treatment, bacteria were, after photosensitization, exposed to 350 pulses of HPPL (UV light dose = 0·023 J cm^?2). Fluorescence measurements were performed to evaluate optimal time for cell–Hyp interaction. Results indicate that Hyp tends to bind both Listeria and Salmonella. After photosensitization treatment, Listeria population was reduced 7 log, whereas Salmonella was inactivated just 1 log. Electron photomicrograps of Salmonella and Listeria confirmed that photosensitization induced total collapse of the Listeria cell wall, but not that of Salmonella. After combined photosensitization–HPPL treatment, the population of Listeria was diminished by 7 log and Salmonella by 6·7 log. Conclusions: Listeria can be effectively inactivated by Hyp‐based photosensitization (7 log), whereas Salmonella is more resistant to photosensitization and can be inactivated just by 1 log in vitro. Combined treatment of photosensitization and pulsed light inactivates effectively (6·7–7 log) both the Gram‐positive and the more resistant to photosensitization Gram‐negative bacteria. Significance and Impact of the Study: A new approach to combat Gram‐positive and Gram‐negative bacteria is proposed, combining photosensitization with high power pulsed light.     

Co-translational incorporation of trans-4-hydroxyproline into recombinant proteins in bacteria

Trans-4-hydroxyproline (Hyp) in eukaryotic proteins arises from post-translational modification of proline residues. Because the modification enzyme is not present in prokaryotes, no natural means exists to incorporate Hyp into proteins synthesized in Escherichia coli. We show here that under appropriate culture conditions Hyp is incorporated co-translationally directly at proline codons in genes expressed in E. coli. The use of Hyp by E. coli protein synthesis machinery under typical culture conditions is not adequate to support protein synthesis; however, intracellular concentrations of Hyp sufficient to compensate for the poor use are achieved in media with hyperosmotic sodium chloride concentrations. Hyp incorporation was demonstrated in several recombinant proteins including human Type I collagen polypeptides. A fragment of the human collagen Type I (alpha1) polypeptide with global Hyp for Pro substitution forms a triple helix. Our results demonstrate a remarkable pliancy in the biosynthetic apparatus of bacteria that may be used more generally to incorporate novel amino acids into recombinant proteins.