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The ubiquitination of the hypoxia-inducible factor-1α (HIF-1α) is mediated by interacting with the von Hippel-Lindau protein (VHL), and is associated with cancer, chronic anemia, and ischemia. VHL, an E3 ligase, has been reported to degrade HIF-1 for decades, however, there are few successful inhibitors currently. Poor understanding of the binding pocket and a lack of in-depth exploration of the interactions between two proteins are the main reasons. Hence, we developed an effective strategy to identify and design new inhibitors for protein-protein interaction targets. The hydroxyproline (Hyp564) of HIF-1α contributed the key interaction between HIF-1α and VHL. In this study, detailed information of the binding pocket were explored by alanine scanning, site-directed mutagenesis and molecular dynamics simulations. Interestingly, we found the interaction(s) between Y565 and H110 played a key role in the binding of VHL/HIF-1α. Based on the interactions, 8 derivates of VH032, 16a-h, were synthesized by introducing various groups bounded to H110. Further assay on protein and cellular level exhibited that 16a-h accessed higher binding affinity to VHL and markable or modest improvement in stabilization of HIF-1α or HIF-1α-OH in HeLa cells. Our work provides a new orientation for the modification or design of VHL/HIF-1α protein-protein interaction inhibitors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Despite the success of imatinib in CML therapy through Bcr-Abl inhibition, acquired drug resistance occurs over time in patients. In particular, the resistance caused by T315I mutation remains a challenge in clinic. Herein, we embarked on a structural optimization campaign aiming at discovery of novel Bcr-Abl inhibitors toward T315I mutant based on previously reported dibenzoylpiperazin derivatives. We proposed that incorporation of flexible linker could achieve potent inhibition of Bcr-Abl T315I by avoiding steric clash with bulky sidechain of Ile315. A library of 28 compounds with amino acids as linker has been developed and evaluated. Among them, compound AA2 displayed the most potent activity against Bcr-Abl WT and Bcr-Abl T315I , as well as toward Bcr-Abl driven K562 and K562R cells. Further investigations indicated that AA2 could induce apoptosis of K562 cells and down regulate phosphorylation of Bcr-Abl. In summary, the compounds with amino acid as novel flexible linker exhibited certain antitumor activities, providing valuable hints for the discovery of novel Bcr-Abl inhibitors to overcome T315I mutant resistance, and AA2 could be considered as a candidate for further optimization., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Commonly used aquatic feed naturally contains low-level or no hydroxyproline (Hyp). This study was conducted to evaluate the effects of dietary Hyp inclusion on growth performance, body composition, amino acid profiles, blood biochemistry, and the expression of target of rapamycin (TOR) pathway-related genes in juvenile Nibea diacanthus. Fish with similar size (initial body weight, 133.00 ± 2.14 g) were fed six isonitrogenous and isolipidic practical diets supplemented with graded levels of Hyp (0, 5, 10, 15, 20, and 25 g kg -1 of dry matter) for 8 weeks. The results indicated that growth performance and feed utilization were improved with increased levels of dietary Hyp (P < 0.05), and the optimum amount of dietary Hyp estimated from SGR as 16.6 g kg -1 . The crude protein of whole body and swim bladder and the amino acid composition of muscle and swim bladder were significantly (P < 0.05) affected by the addition of dietary Hyp, which reflects the important role of feed composition in animal body composition. In addition, the expression levels of mammalian target of rapamycin (TOR) and ribosomal protein S6 kinase1 (S6K1) genes in the liver, muscle, and swim bladder increased with increasing Hyp content of diets, while the mRNA expression level of eukaryotic translation initiation factor 4E-binding protein (4E-BP) gene in these tissues decreased. These results indicated that Hyp improved fish growth and the ability to synthesize proteins, most likely through the TOR pathway. It is suggested that dietary Hyp supplementation is particularly necessary for application in aquatic feed.

The N -methyl-(2S,4R)-trans-4-hydroxy-l-proline-enriched fraction (NMP) from Sideroxylon obtusifolium was evaluated as a neuroprotective agent in the intracerebroventricular (icv) pilocarpine (Pilo) model. To this aim, male mice were subdivided into sham (SO, vehicle), Pilo (300 µg/1 µL icv, followed by the vehicle per os , po ) and NMP-treated groups (Pilo 300 µg/1 µL icv, followed by 100 or 200 mg/kg po ). The treatments started one day after the Pilo injection and continued for 15 days. The effects of NMP were assessed by characterizing the preservation of cognitive function in both the Y-maze and object recognition tests. The hippocampal cell viability was evaluated by Nissl staining. Additional markers of damage were studied-the glial fibrillary acidic protein (GFAP) and the ionized calcium-binding adaptor molecule 1 (Iba-1) expression using, respectively, immunofluorescence and western blot analyses. We also performed molecular docking experiments revealing that NMP binds to the γ-aminobutyric acid (GABA) transporter 1 (GAT1). GAT1 expression in the hippocampus was also characterized. Pilo induced cognitive deficits, cell damage, increased GFAP, Iba-1, and GAT1 expression in the hippocampus. These alterations were prevented, especially by the higher NMP dose. These data highlight NMP as a promising candidate for the protection of the hippocampus, as shown by the icv Pilo model.

Oxaceprol is well-defined therapeutic agent as an atypical inhibitor of inflammation in osteoarthritis. In the present study, we aimed to develop and characterize oxaceprol-loaded poly-lactide-co-glycolide (PLGA) nanoparticles for intra-articular administration in osteoarthritis. PLGA nanoparticles were prepared by double-emulsion solvent evaporation method. Meanwhile, a straightforward and generally applicable high performance liquid chromatography method was developed, and validated for the first time for the quantification of oxaceprol. To examine the drug carrying capacity of nanoparticles, varying amount of oxaceprol was entrapped into a constant amount of polymer matrix. Moreover, the efficacy of drug amount on nanoparticle characteristics such as particle size, zeta potential, morphology, drug entrapment, and in vitro drug release was investigated. Nanoparticle sizes were between 229 and 509 nm for different amount of oxaceprol with spherical smooth morphology. Encapsulation efficiency ranged between 39.73 and 63.83% by decreasing oxaceprol amount. The results of Fourier transform infrared and DSC showed absence of interaction between oxaceprol and PLGA. The in vitro drug release from these nanoparticles showed a sustained release of oxaceprol over 30 days. According to cell culture studies, oxaceprol-loaded nanoparticles had no cytotoxicity with high biocompatibility. This study was the first step of developing an intra-articular system in the treatment of osteoarthritis for the controlled release of oxaceprol. Our findings showed that these nanoparticles can be beneficial for an effective treatment of osteoarthritis avoiding side effects associated with oral administration., (© 2020 Wiley Periodicals, Inc.)

Scope: Inflammatory bowel disease (IBD) is a chronic disease of gastrointestinal tract in which oxidative stress and overactivation of inflammatory response are implicated. The aim of the present study is to test the hypothesis that hydroxyproline (Hyp), an amino acid with an antioxidative property, attenuates dextran sulfate sodium (DSS)-induced colitis in mice., Methods and Results: Male C57BL/6 mice supplemented with or without 1% Hyp are subjected to 2.5% DSS in drinking water to induce colitis. Hyp attenuates the severity of colitis as evidenced by reduced disease activity index scores, decreased myeloperoxidase activity, histological damage, and apoptosis. Furthermore, DSS-induced increases in reactive oxygen species accumulation, TNF-α and IL-6 secretion, and malonyldialdehyde activity and a decrease in reduced glutathione in the colon are ameliorated by Hyp. The enhanced phosphorylation of STAT3 and NF-κB following DSS administration is mitigated by Hyp, which is also observed in LPS-treated RAW264.7 macrophages. Moreover, the inhibitory effect of Hyp on IL-6 expression is mainly mediated by the NF-κB signaling, because the induction of STAT3 and IL-6 by LPS is markedly reversed by Bay11-7085, a specific inhibitor NF-κB., Conclusion: In summary, Hyp is a critical nutrient with an ability to attenuate DSS-induced colonic damage in mice. This beneficial effect of Hyp is partially mediated by inhibiting the NF-κB/IL-6 signaling and the restoration of redox homeostasis., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Proline (Pro) accumulation protects plant cell under abiotic stress. Hydroxyproline (Hyp) as selection agent is a toxic analog of proline and promotes Pro overaccumulation. In this study, Chardonnay calli were firstly irradiated with different dosages of 60 Co and then cultured on a Hyp-added medium. Finally, some stable hydroxyproline-resistant (HR) calli were obtained. When calli were cultured on 4 mM Hyp medium for 7 days, intracellular Pro content of the HR calli was five times higher than that detected in the normal calli. The regeneration of HR calli into plantlets was much slower than that of normal ones. When cultured on woody plant medium (WPM) containing 10 mM NaCl for 14 days, HR plantlets still grew well with lower Pro than withered normal plantlets. qRT-PCR results of Pro biosynthesis-related genes in HR plantlets showed that three genes VvP5CS, VvOAT, and VvP5CDH were conducive for Pro accumulation. These results confirmed that HR plantlets acquired salt tolerance ability. We prospect that this procedure to obtain salt-tolerant plants may be valuable to breed programs and improve grapevine genotypes with increased tolerance to salt and other abiotic stresses.

Aim: This study was conducted to estimate the aminoacid levels in the vitreous of patients with proliferative diabetic retinopathy, and to correlate it with the adiponectin levels. Secondly to test if these amino acids can alter or induce adiponectin levels and its related factors in retinal cells like pericyte as an in vitro model., Methods: All human studies were done as per declaration of Helsinki with institutional approval and after obtaining consent from participating individuals. The vitreous amino acids were estimated in PDR (Proliferative diabetic retinopathy) and MH (Macular Hole) as disease control using HPLC. Bovine retinal pericytes (BRP) were cultured in DMEM/F12 medium and treated with 0.5 mM of any one of the individual amino acids (proline, hydroxyproline, phenylalanine, alanine, serine, glycine, lysine, isoleucine or valine) along with 100 nM insulin for 14 days in high glucose (25 mM) condition. The mRNA expression profile of adipogenic markers (such as Pref1, APN, ZAG and PPARγ), angiogenic markers (VEGF, MMP-2 and MMP-9, TGF-β) and antioxidant markers (Nrf2 and UCP-2) were evaluated by qPCR. Adipogenesis was further confirmed by adipogenesis assay, secretion of adiponectin in medium and triglyceride accumulation by Oil red O staining in Bovine retinal pericytes., Results: Amino acids valine (p < 0.004), isoleucine (p < 0.0007), leucine (p < 0.022), serine (p < 0.0007), glycine (p < 0.001), alanine (p < 0.017), phenylalanine (p < 0.013), and lysine (p < 0.001) were significantly elevated in the vitreous of PDR group (n = 30) when compared to macular hole (n = 20). There was a significant positive correlation between serine (p < 0.021), alanine (p < 0.00016), phenylalanine (p < 0.04), isoleucine (p < 0.023), leucine (p < 0.043), and lysine (p < 0.026) with adiponectin level in the vitreous. The amino acids hydroxyproline, proline, lysine, glycine and alanine induced the triglyceride accumulation and expression of Adiponectin. VEGF and MMP-9 expression was decreased with all the amino acids treated and PEDF was significantly increased with phenylalanine treatment. TGFβ mRNA expression showed a significant decrease with proline, alanine, glycine, lysine and isoleucine. The Nrf2 expression was significantly increased in alanine and serine when compared to control. The UCP-2 gene showed a significant increase in proline and lysine treatment., Discussion and Conclusion: Our results suggest that amino acids hydroxyproline, proline, lysine, glycine and alanine which are elevated in the PDR vitreous show a tendency to induce adipogenic effects in retinal pericytes by triggering the accumulation of triglycerides and adiponectin. Hence we hypothesize that these aminoacids when elevated along with insulin and glucose can induce metabolic changes in pericytes. The functional implications of these changes tend to be protective as it increases the antioxidant potential and decreases the angiogenesis markers which are potentially pathogenic., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Substrate reduction therapy with analogs reduces the accumulation of substrates by inhibiting the metabolic pathways involved in their biosynthesis, providing new treatment options for patients with primary hyperoxalurias (PHs) that often progress to end-stage renal disease (ESRD). This research aims to evaluate the inhibition efficacy of Hydroxy-L-proline (HYP) analogs against calcium oxalate (CaOx) crystal formation in the Drosophila Melanogaster (D. Melanogaster) by comparing them with Pyridoxine (Vitamin B6)., Methods: Three stocks of Drosophila Melanogaster (W 118 , CG3926 RNAi, and Act5C-GAL4/CyO) were utilized. Two stocks (CG3926 RNAi and Act5C-GAL4 /CyO) were crossed to generate the Act5C > dAGXT RNAi recombinant line (F 1 generation) of D. Melanogaster which was used to compare the efficacy of Hydroxy-L-proline (HYP) analogs inhibiting CaOx crystal formation with Vitamin B 6 as the traditional therapy for primary hyperoxaluria., Results: Nephrolithiasis model was successfully constructed by downregulating the function of the dAGXT gene in D. Melanogaster (P-Value = 0.0045). Furthermore, the efficacy of Hydroxy-L-proline (HYP) analogs against CaOx crystal formation was demonstrated in vivo using D. Melanogaster model; the results showed that these L-Proline analogs were better in inhibiting stone formation at very low concentrations than Vitamin B 6 (IC 50  = 0.6 and 1.8% for standard and dietary salt growth medium respectively) compared to N-acetyl-L-Hydroxyproline (IC 50  = 0.1% for both standard and dietary salt growth medium) and Baclofen (IC 50  = 0.06 and 0.1% for standard and dietary salt growth medium respectively). Analysis of variance (ANOVA) also showed that Hydroxy-L-proline (HYP) analogs were better alternatives for CaOx inhibition at very low concentration especially when both genetics and environmental factors are intertwined (p < 0.0008) for the dietary salt growth medium and (P < 0.063) for standard growth medium., Conclusion: Addition of Hydroxy-L-Proline analogs to growth medium resulted in the reduction of CaOx crystals formation. These analogs show promise as potential inhibitors for oxalate reduction in Primary Hyperoxaluria.

Chemical cell-surface engineering is a tool for modifying and altering cellular functions. Herein, we report the introduction of an antibiotic phenotype to the green alga Chlamydomonas reinhardtii by chemically modifying its cell surface. Flow cytometry and confocal microscopy studies demonstrated that a hybrid of the antibiotic vancomycin and a 4-hydroxyproline oligomer binds reversibly to the cell wall without affecting the viability or motility of the cells. The modified cells were used to inhibit bacterial growth of Gram-positive Bacillus subtilis cultures. Delivery of the antibiotic from the microalgae to the bacterial cells was verified by microscopy. Our studies provide compelling evidence that 1) chemical surface engineering constitutes a useful tool for the introduction of new, previously unknown functionality, and 2) living microalgae can serve as new platforms for drug delivery., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: This study investigated the effects of an amino acid mixture containing arginine, glutamine, and β-hydroxy-β-methyl butyrate on secondary healing of ischemic wounds in a rat model (N = 18)., Methods: After the formation of a bipediculated flap on each rat, 2 full-thickness excisional skin wounds (2 × 2 cm) were created on every flap. The rats were then randomized into the control and treatment groups. Every rat received standardized rat food throughout the study. The rats in the treatment group were administered an extra 200 mg/kg of L-arginine, 200 mg/kg of L-glutamine, and 40 mg/kg of β-hydroxy-β-methyl butyrate per day. Wound sizes were measured on days 0, 4, 10, and 14. The rats were sacrificed, and the wounds were excised for biochemical and histologic examination on the 14th day., Results: As compared with the control group, the treatment group's wound sizes were significantly smaller on days 10 and 14 ( P < .001), as was its inflammatory cell accumulation score ( P = .008). There was no significant difference between the 2 groups in collagen accumulation ( P = .340), granulation tissue maturation ( P = .161), angiogenesis ( P = .387), or reepithelialization ( P = .190) and no significant difference between hydroxyproline concentrations in wounds ( P = .287)., Discussion: This amino acid combination seems to have a positive impact on the secondary healing of experimental ischemic wounds when introduced as a supplement to the standard diet, and the reduction in the inflammatory process appears to play a role in this effect.

Conantokins are ~20-amino acid peptides present in predatory marine snail venoms that function as allosteric antagonists of ion channels of the N-methyl-d-aspartate receptor (NMDAR). These peptides possess a high percentage of post-/co-translationally modified amino acids, particularly γ-carboxyglutamate (Gla). Appropriately spaced Gla residues allow binding of functional divalent cations, which induces end-to-end α-helices in many conantokins. A smaller number of these peptides additionally contain 4-hydroxyproline (Hyp). Hyp should prevent adoption of the metal ion-induced full α-helix, with unknown functional consequences. To address this disparity, as well as the role of Hyp in conantokins, we have solved the high resolution three-dimensional solution structure of a Gla/Hyp-containing 18-residue conantokin, conRl-B, by high field NMR spectroscopy. We show that Hyp(10) disrupts only a small region of the α-helix of the Mn(2+)·peptide complex, which displays cation-induced α-helices on each terminus of the peptide. The function of conRl-B was examined by measuring its inhibition of NMDA/Gly-mediated current through NMDAR ion channels in mouse cortical neurons. The conRl-B displays high inhibitory selectivity for subclasses of NMDARs that contain the functionally important GluN2B subunit. Replacement of Hyp(10) with N(8)Q results in a Mg(2+)-complexed end-to-end α-helix, accompanied by attenuation of NMDAR inhibitory activity. However, replacement of Hyp(10) with Pro(10) allowed the resulting peptide to retain its inhibitory property but diminished its GluN2B specificity. Thus, these modified amino acids, in specific peptide backbones, play critical roles in their subunit-selective inhibition of NMDAR ion channels, a finding that can be employed to design NMDAR antagonists that function at ion channels of distinct NMDAR subclasses., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

In order to enlarge the potential resources of drought-tolerant peanuts, we conducted in vitro mutagenesis with Pingyangmycin (PYM) as the mutagen as well as directed screening on a medium supplemented with Hydroxyproline (HYP). After being extracted from mature seeds (cv. Huayu 20), the embryonic leaflets were cultured on somatic embryogenesis-induction medium with 4 mg/L PYM and the generated embryos were successively transferred to a germination medium with 4 and then 8 mmol/L HYP to screen HYP-tolerant plantlets. After that, these plantlets were grafted and transplanted to the experimental field. In the next generation, all seeds were sown in the field, and phenotype variation and trait segregation can be observed in most of the offspring (M2 generation). The M3 generation individuals were subjected to drought stress at the seedling stages. The activities of SOD and POD were substantially increased in eight offspring of 11 HYP-tolerant, regenerated plants than in their mutagenic parents. To determine the correlation between mutant phenotypes and genomic modification, we carried out a comparison of the DNA polymorphisms between the mutagenic parents and 13 M3 generation individuals from different HYP-tolerant, regenerated plants with SSR primers. Results showed that most mutants and parent plants had signs of polymorphisms. Under drought stress, some M3 generation individuals of 10 original HYP-tolerant, regenerated plants produced more pods than the mutagenic parent; twenty individuals among them produced >60 g pods/plant. M4-generation seeds were tested for quality characteristics by Near Infrared Spectroscopy (NIS) and nine individuals with higher protein content (>30%) and 21 individuals with higher oil content (>58%) were screened. We concluded that the use of PYM-based in vitro mutagenesis in combination with directed screening with HYP is effective for the creation of potential drought-tolerant mutants of peanut.

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.

Hydroxyproline-rich glycoproteins (HRGPs) are plant cell wall proteins related to plant growth and development, and extensins (EXTs) are a subfamily of HRGPs. In this study, the function of HRGPs, especially EXTs, was investigated in the pollen tube and style cell growth of Nicotiana tabacum L. By using the techniques of protein blot and immunohistochemistry, the JIM20-recognized epitopes of EXTs were abundantly expressed in vivo for pollen tubes and transmitting tissue. A hydroxyproline synthesis inhibitor, 3,4-dehydro-l-proline (3,4-DHP), was used to investigate the functions of HRGPs. The addition of 3,4-DHP decreased the speed of pollen tube growth and shortened the length of style. Moreover, the hydroxyproline assay and JIM20 immunolocalization confirmed that 3,4-DHP treatment reduced the level of hydroxyproline and EXTs in the treated styles, respectively. These results indicate that HRGPs, most likely EXTs, may play important roles in the pollen tube and style cell growth., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

In humans and rodents, dietary hydroxyproline (hyp) and oxalate intake affect urinary oxalate (Uox) excretion. Whether Uox excretion occurs in cats was tested by feeding diets containing low oxalate (13 mg/100 g DM) with high (Hhyp-Lox), moderate (Mhyp-Lox), and low hyp (Lhyp-Lox) concentrations (3.8, 2.0, and 0.2 g/100 g DM, respectively) and low hyp with high oxalate (93 mg/100 g DM; Lhyp-Hox) to 8 adult female cats in a 48-d study using a Latin square design. Cats were randomly allocated to one of the four 12-d treatment periods and fed according to individual energy needs. Feces and urine were collected quantitatively using modified litter boxes during the final 5 d of each period. Feces were analyzed for oxalate and Ca, and urine was analyzed for specific density, pH, oxalate, Ca, P, Mg, Na, K, ammonia, citrate, urate, sulfate, and creatinine. Increasing hyp intake (0.2, 2.0, and 3.8 g/100 g DM) resulted in increased Uox excretion (Lhyp-Lox vs. Mhyp-Lox vs. Hhyp-Lox; P < 0.05), and the linear dose-response equation was Uox (mg/d) = 5.62 + 2.10 × g hyp intake/d (r(2) = 0.56; P < 0.001). Increasing oxalate intake from 13 to 93 mg/100 g DM did not affect Uox excretion but resulted in an increase in fecal oxalate output (P < 0.001) and positive oxalate balance (32.20 ± 2.06 mg/d). The results indicate that the intestinal absorption of the supplemental oxalate, and thereby its contribution to Uox, was low (5.90% ± 5.24%). Relevant increases in endogenous Uox excretion were achieved by increasing dietary hyp intake. The hyp-containing protein sources should be minimized in Ca oxalate urolith preventative diets until their effect on Uox excretion is tested. The oxalate content (up to 93 mg/100 g DM) in a diet with moderate Ca content does not contribute to Uox content.

We examined the effects of oral L-hydroxyproline (Hyp) on collagen in the body. After 2 weeks' administration of Hyp (0.5 or 1 g/kg) to F344 male rats, the soluble collagen content of the skin had increased, and the serum concentration of collagen peptides was correlated with the skin content of soluble collagen. This result suggests that oral Hyp augmented collagen metabolism.

Current clinically used chemotherapeutic platinum drugs can trigger severe toxic effects. To develop a model system for the evaluation of the therapeutic efficacy and the toxic effects of new platinum agents, we have synthesized a new compound N-[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxylhex-1-yl]-L-hydroxyproline dichloroplatinum(ii) (PHDP), compared its in vitro anti-proliferation activity, in vivo anti-tumor activity and safety to those of oxaliplatin, and correlated all these biological actions with the platinum occurring in the spleen, kidney, heart, brain, blood, tumor tissue, urine and faeces of the treated mice. We explored the atomic absorption based determinations of the platinum which occurred in the spleen, kidney, heart, brain, blood, tumor tissue, urine and faeces and constitute a model system that can be generally used in the investigation of the novel platinum agents.

A series of new HIV-1 protease inhibitors with the hydroxyethylamine core and different hydroxyprolinamide P2 ligands were designed and synthesized. Variation of substitutions at the P2 significantly affected the enzyme inhibitory potency of the inhibitors. Compounds 2a and 2d showed excellent enzyme inhibitory activity with IC(50) values in the nanomolar range. An active site binding model for inhibitors 2a and 2d was suggested based upon the computational-docking results of the ligand with HIV-1 protease. This model offers molecular insights regarding ligand-binding site interactions of the hydroxyprolinamide-derived novel P2-ligand., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

A bacterial strain, designated DR-834 and producing immunostimulatory activities to carp (Cyprinus carpio), was isolated from Qinghai-Tibetan Plateau permafrost soil. Cultural characteristic studies suggested that this strain belongs to the genus Bacillus. The nucleotide sequence of the 16S rRNA gene of strain DR-834 exhibited close similarity (99%) with the 16S rRNA gene of Bacillus simplex. Two compounds showing potent activity were isolated from secondary metabolites of the strain through bioassay-guided isolation techniques and identified by spectral data (infrared, nuclear magnetic resonance and mass spectrometry) as: (1) 4-trans-hydroxy-l-proline and (2) cyclo-(l-Pro-Gly)(2). They were found to be significantly increased the selected innate immune function parameters, serum SOD activity, serum lysozyme activity, serum bactericidal activity, superoxide anion production and phagocytic activity by isolated blood leucocytes. The effects of two compounds on immune-related genes expression were further investigated. The outcomes of real-time quantitative polymerase chain reaction (RQ-PCR) proved that the transcribing level of interleukin 1beta (IL-1beta) and inducible nitric oxide synthase (iNOS) mRNA in the blood have been augmented by 4-trans-hydroxy-l-proline and cyclo-(l-Pro-Gly)(2). Compounds 1 and 2 administration the challenge with live Aeromonas hydrophila decreased the percentage mortality in the experimental groups with the consequence increase in relative percent survival (RPS) values. Compound 2 produced the highest protection with the RPS values of 87.50, 77.78, 55.56 and 55.56 after 1, 2, 3 and 4 weeks, respectively. The study indicates that the isolated compounds could be positively influence the immune response and protect the heath status of carp against A. hydrophila infection., (Crown Copyright 2010. Published by Elsevier Ltd. All rights reserved.)

Purpose: This study addressed the question of whether the collagen metabolism modulator cis-4-Hydroxy-L-proline (CHP) is applicable for potential use as a therapeutic inhibitor of pancreatic carcinoma cell growth., Methods: Cell proliferation, as well as quantification of apoptosis of murine Panc02 cells, was assessed after CHP treatment. Supplementary, the effect of CHP on tumor growth was examined in the subcutaneous Panc02 model in vivo. Mice received daily intraperitoneal injections of CHP (300, 400, and 500 mg/kg bw). In addition to the assessment of systemic parameters (blood count, enzyme activities), histology (HE) and immunohistochemistry (Ki-67) were performed from resected tumor specimens., Results: Like reduction of metabolic activity, CHP also induced inhibition of cell growth in a dose-dependent manner, with however only slight increases in apoptosis. In vivo treatment of Panc02 tumors with CHP resulted in pronounced delay of tumor growth and decreases in tumor cell proliferation. Moreover, these effects were accompanied by a massive systemic leukocytosis as well increased leukocyte infiltration into the tumors subsequent to CHP therapy., Conclusions: CHP inhibits the proliferation of Panc02 tumor cells in a dose-dependent manner both in vitro and in vivo. Our presented data show that modulation of the collagen metabolism is an interesting strategy for treatment of pancreatic carcinoma.

Background: The most important function of collagen and elastin is to induce several mechanical parameters which are known to play a dominant role in governing mechanical properties of the blood vessels. The aortic tissue of rabbit is one of the important sources of collagen and elastin. The effects of high fat diet (HFD) on the hydroxyproline (Hyp) fractions in serum and aortic tissues of rabbits and collagen content in the aortic tissues of rabbits have not been documented before. The present study was undertaken to investigate the changes in Hyp fractions in serum and aortic tissues of rabbits and collagen content in the aortic tissues of rabbits during the progression of atherosclerosis. The atherosclerotic model used in this study was the New Zealand white rabbit (male; 12 weeks old). Twenty five rabbits were individually caged, and divided into control group (NOR; n = 10) and HFD group (CHO; n = 15). The control group was fed (100 g/day) of normal (NOR) diet for a period of 15 weeks. The HFD group was fed normal diet supplemented with 1.0% cholesterol plus 1.0% olive oil (100 g/day) for the same period of time., Results: We found that the TC, LDLC, and TG (mg/dl) were significantly (p < 0.001) increased in HFD rabbits compared with control rabbits with percentage normalized changes of 1198%, 1591%, and 710%, respectively. The peptide-bound Hyp in the serum was significantly (P < 0.05) increased in HFD rabbits compared with control rabbits with percentage normalized change of 517% while it significantly (P < 0.01) decreased in aortic tissues of HFD rabbits compared with control rabbits with percentage normalized change of 65%. The protein-bound Hyp in the serum was significantly (P < 0.01) increased in HFD rabbits compared with control rabbits with percentage normalized change of 100%; the protein-bound Hyp in the aortic tissues of control rabbits was 235.30 +/- 55.14 (Mean +/- SD) while it was not detectable (ND) in HFD rabbits. Total serum Hyp showed no significant (P < 0.05) change in HFD rabbits compared with control rabbits while it was significantly (P < 0.05) decreased in aortic tissues of HFD rabbits compared with control rabbits with percentage normalized change of 73%. The total collagen was significantly (p < 0.01) decreased in aortic tissues of HFD rabbits compared with control rabbits with percentage normalized change of 73% which was supported by histological study., Conclusions: These results suggest that percentage decrease in various Hyp fractions in aortic tissue of HFD rabbits are closely related to percentage decrease of collagen content in aortic tissues of HFD rabbits. These results also suggest that it may be possible to use the changes in various Hyp fractions in aortic tissues of rabbits as an important risk factor during the progression of atherosclerosis.

Hydroxyproline-rich proteins in plants offer a source of carbon and nitrogen to soil-dwelling microorganisms in the form of root exudates and decaying organic matter. This report describes an ABC-type transport system dedicated to the uptake of hydroxyproline in the legume endosymbiont Sinorhizobium meliloti. We have designated genes involved in hydroxyproline metabolism as hyp genes and show that an S. meliloti strain lacking putative transport genes (DeltahypMNPQ) is unable to grow with or transport trans-4-hydroxy-l-proline when this compound is available as a sole source of carbon. Expression of hypM is upregulated in the presence of trans-4-hydroxy-l-proline and cis-4-hydroxy-d-proline, as modulated by a repressor (HypR) of the GntR/FadR subfamily. Although alfalfa root nodules contain hydroxyproline-rich proteins, we demonstrate that the transport system is not highly expressed in nodules, suggesting that bacteroids are not exposed to high levels of free hydroxyproline in planta. In addition to hypMNPQ, we report that S. meliloti encodes a second independent mechanism that enables transport of trans-4-hydroxy-l-proline. This secondary transport mechanism is induced in proline-grown cells and likely entails a system involved in l-proline uptake. This study represents the first genetic description of a prokaryotic hydroxyproline transport system, and the ability to metabolize hydroxyproline may contribute significantly toward the ecological success of plant-associated bacteria such as the rhizobia.

A combination of spectroscopic, calorimetric, and microscopic studies to understand the effect of hydroxyproline on the thermal stability, conformation, biological activity, and aggregation of proteins has been investigated. Significantly increased protein stability and suppression of aggregation is achieved in the presence of hydroxyproline. For example, exceptional increase in the thermal stability of lysozyme up to 26.4 degrees C and myoglobin up to 31.8 degrees C is obtained in the presence of hydroxyproline. The increased thermal stability of the proteins is observed to be accompanied with significant rise of the catalytic activity. Hydroxyproline is observed to prevent lysozyme fibril formation in vitro. Fluorescence and circular dichroism studies indicate induction of tertiary structures of the studied proteins in the presence of hydroxyproline. Preferential hydration of the native state is found to be crucial for the mechanism of protein stabilization by hydroxyproline. We compared the effect of hydroxyproline to that of proline and observed similar increase in the activity and suppression of protein aggregation. The results demonstrate the use of hydroxyproline as a protein stabilizer and in the prevention of protein aggregation and fibril formation.

The effects of an intravenous hydroxyproline load on endogenous oxalogenesis were compared in rats fed a standard diet or a vitamin B6-deficient diet. Twelve male Wistar rats were randomized to two groups and were fed either a standard diet (control group) or a vitamin B6-deficient diet for 3 weeks. Then the animals were intravenously administered 100 mg (762.6 micromol)/ml hydroxyproline. In the control group, infusion of hydroxyproline increased the 5-h urinary oxalate and glycolate excretion above baseline to 0.27% (2.02 +/- 1.11 micromol) and 0.32% (2.43 +/- 1.60 micromol) of the administered dose (mol/mol), while it was respectively 2.01% (15.24 +/- 2.13 micromol) and 0.00% (-0.02 +/- 0.19 micromol) of the dose in the vitamin B6-deficient group. Therefore, vitamin B6 deficiency augmented endogenous synthesis of oxalate from hydroxyproline by 7.56-fold (15.24/2.02) compared with that in the control group. Urinary citrate excretion was significantly lower at baseline and all other times in the vitamin B6-deficient group compared with the control group. In conclusions, L-hydroxyproline loading augmented endogenous oxalogenesis in the vitamin B6-deficient group without causing hyperglycolic aciduria, and also led to significant hypocitraturia. These findings suggest that hydroxyproline is not metabolized to oxalate via glycolate, but rather via the 4-hydroxyglutamate to glyoxylate pathway (usually requiring vitamin B6-dependent enzymes) even in the presence of vitamin B6 deficiency.

Aim: To investigate the biological effects of cis-hydroxyproline (CHP) on the rat pancreatic carcinoma cell line DSL6A, and to examine the underlying molecular mechanisms., Methods: The effect of CHP on DSL6A cell proliferation was assessed by using BrdU incorporation. The expression of focal adhesion kinase (FAK) was characterized by Western blotting and immunofluorescence. Induction of endoplasmic reticulum (ER) stress was investigated by using RT-PCR and Western blotting for the glucose-related protein-78 (GRP78) and growth arrest and DNA inducible gene (GADD153). Cell viability was determined through measuring the metabolic activity based on the reduction potential of DSL6A cells. Apoptosis was analyzed by detection of caspase-3 activation and cleavage of poly(ADP-ribose) polymerase (PARP) as well as DNA laddering., Results: In addition to inhibition of proliferation, incubation with CHP induced proteolytic cleavage of FAK and a delocalisation of the enzyme from focal adhesions, followed by a loss of cell adherence. Simultaneously, we could show an increased expression of GRP78 and GADD153, indicating a CHP-mediated activation of the ER stress cascade in the DSL6A cell line. Prolonged incubation of DSL6A cells with CHP finally resulted in apoptotic cell death. Beside L-proline, the inhibition of intracellular proteolysis by addition of a broad spectrum protease inhibitor could abolish the effects of CHP on cellular functions and the molecular processes. In contrast, impeding the activity of apoptosis-executing caspases had no influence on CHP-mediated cell damage., Conclusion: Our data suggest that the initiation of ER stress machinery by CHP leads to an activation of intracellular proteolytic processes, including caspase-independent FAK degradation, resulting in damaging pancreatic carcinoma cells.

Effects of adenosine on the excitability of supraoptic nucleus neurons were investigated in whole cell patch-clamp experiments conducted in horizontal slices of rat hypothalamus. Adenosine (10-100 muM) inhibited all neurons tested by reducing or abolishing spontaneous or evoked discharge. Large hyperpolarizations were seen, averaging -6.08 +/- 0.83 mV below resting membrane potential, and action potential durations were significantly reduced by 134 +/- 41 mus in the presence of 100 muM adenosine. The A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 1 muM) blocked these effects, whereas the A(1) agonists N(6)-cyclopentyladenosine (CPA) and N(6)-cyclohexyladenosine (CHA) mimicked the actions of adenosine. A(2) receptor contributions to excitability were assessed by application of an A(2) agonist, carboxamidoadenosine (CPCA). This resulted in membrane depolarizations (3.56 +/- 0.65 mV) and maintenance of firing. The presence of endogenous adenosine in the slice was revealed by both the application of the adenosine uptake inhibitor dilazep (1-100 muM), which resulted in a strong inhibition of firing activity, and the application of DPCPX, which induced firing in cells silenced by negative current injection. We tested for postsynaptic actions of adenosine by blocking G protein activation via GDP-beta-S infusion into recorded neurons. Under these conditions, the adenosinergic inhibition of firing and reduction of spike duration were blocked, suggesting the effects were mediated by postsynaptic adenosine receptors. That the effects on excitability could be due to direct activation of adenosine A(1) receptors on supraoptic neurons was further explored immunocytochemically via the co-labeling of magnocellular neurons with polyclonal antibodies raised against the A(1) receptors. It is concluded that adenosine, acting at postsynaptic A(1) receptors, exhibits a powerful inhibitory influence on supraoptic magnocellular activity and is an important endogenous regulator of magnocellular neuroendocrine function.

Purpose: This work was carried out to follow up the healing of full-thickness incision wounds opened on the back skin of rabbits in order to gain insight into the periodical correlation among such factors as ascorbic acid, collagen (hydroxyproline), the zinc content, and tensile strength of wound tissue. The need to provide vitamin C or zinc supplements after such wound incisions is also discussed., Methods: Full-thickness incision wounds and the ascorbic acid and hydroxyproline levels were measured in 24 rabbits by spectrophotometric methods on day 0 and on the 3rd, 5th, 7th, and 15th days after operation. The tensile strength was measured by a polygraph using a force displacement transducer. The zinc levels of the wounds were measured by atomic absorption spectrophotometry., Results: The zinc and hydroxyproline levels reached the peak levels on the 5th day, but the tensile strength of wound increased sharply on the 7th day after wounding while the zinc levels did not change. These results indicated that in the first 7 days of wound healing, high levels of ascorbic acid, hydroxyproline, and zinc cumulation occurred in the wound tissue, and the tensile strength reached its highest level on the 15th day without any supplementation., Conclusion: The supplementation of zinc and/or ascorbic acid should therefore be given just at the beginning of the wounding period, especially if there is deficiency of these nutrients.

The collagen triple helix is composed of three polypeptide strands, each with a sequence of repeating (Xaa-Yaa-Gly) triplets. In these triplets, Xaa and Yaa are often tertiary amides: L-proline (Pro) and 4(R)-hydroxy-L-proline (Hyp). To determine the contribution of tertiary amides to triple-helical stability, Pro and Hyp were replaced in synthetic collagen mimics with a non-natural acyclic tertiary amide: N-methyl-L-alanine (meAla). Replacing a Pro or Hyp residue with meAla decreases triple-helical stability. Ramachandran analysis indicates that meAla residues prefer to adopt straight phi and psi angles that are dissimilar from those of the Pro and Hyp residues in the collagen triple helix. Replacement with meAla decreases triple-helical stability more than does replacement with Ala. All of the peptide bonds in triple-helical collagen are in the trans conformation. Although an Ala residue greatly prefers the trans conformation, a meAla residue exists as a nearly equimolar mixture of trans and cis conformers. These findings indicate that the favorable contribution of Pro and Hyp to the conformational stability of collagen triple helices arises from factors other than their being tertiary amides., (Copyright 2001 John Wiley & Sons, Inc. Biopolymers 59: 24-28, 2001)

Oxaceprol (N-acetyl-L-hydroxyproline), an atypical inhibitor of inflammation, is an established drug forjoint disease without serious side-effects. Recent studies have emphasized that oxaceprol has an effect on the microcirculation. Since the exact mechanism of action remains unclear, the aim of our study was to investigate the leukocyte-endothelial cell interactions in oxaceprol-treated mice with antigen-induced arthritis (AiA) using intravital microscopy. In our study, Balb/c mice were allocated to 4 groups (n 7, 8, 8, 8): 2 control groups with saline or oxaceprol and 2 groups of arthritic animals which received saline or oxaceprol (100 mg/kg twice a day intraperitoneally). The severity of arthritis was quantified by the transverse knee joint diameter. For the intravital fluorescence microscopy measurements on day 10 after inducing arthritis, the patella tendon was partily resected to visualize the intraarticular synovial tissue of the knee joint. The number of rolling and adherent leukocytes as well as RBC velocity and functional capillary density (FCD) were quantified in synovial microvessels. Furthermore, leukocyte infiltration was determined in the histological sections with an established score. No significant changes in mean arterial blood pressure or functional capillary density were found in any of the groups. However, the leukocyte rolling fraction and number of leukocytes adherent to the endothelium were increased in postcapillary venules of the synovium in arthritic animals (0.16 to 0.31, 78 cells/mm2 to 220 cells/mm2). In animals with AiA treated with oxaceprol, leukocyte adherence and swelling were significantly reduced in comparison to the arthritic animals treated with saline. Furthermore, the histological score showed less leukocyte infiltration in the oxaceprol treated arthritic animals. Thus, oxaceprol reduces leukocyte adherence in vivo and leukocyte infiltration in mouse AiA, indicating an effect on synovial microcirculation.

The role of fibrillar collagen on myogenic differentiation has previously been studied in tissue culture cell lines but has not been studied in situ. We treated cultured chick and mouse embryos with collagen synthesis inhibitors to determine the role of fibrillar collagen on somitogenesis and on myogenic differentiation in vivo. Stage 12 chick embryos and 8.7 dpc mouse embryos were cultured in control medium or a range of concentrations of the collagen synthesis inhibitors ethyl-3,4-dihydroxybenzoate (EDHB) or cis-hydroxy-proline (CHP). Chick embryos were cultured for 24 h and mouse embryos were cultured for 30 h. Both collagen synthesis inhibitors produced a range of somite abnormalities including formation of fewer and irregular somites in both chick and mouse at high drug concentrations, as well as formation of double somites in EDHB-treated chick embryos. Examination of EDHB-treated mouse embryos by scanning electron microscopy demonstrated a dosage-dependent loss of fibrillar collagen and associated extracellular matrix. Expression of myogenin in EDHB-treated mouse embryos, examined by whole-mount in situ hybridization, was suppressed at higher dosage levels. This study suggests that inhibition of fibrillar collagen production and/or loss of fibrillar collagen in the developing avian and mammalian embryo results in abnormal somite formation and perturbed myogenic differentiation.

With the ever-increasing evidence that the extracellular matrix (ECM) can stimulate tumor growth, it follows that inhibiting the synthesis of tumor-derived stroma may be a potential therapeutic target of cancer progression. The proline analog cis-hydroxyproline (CHP), an inhibitor of collagen deposition, was examined for its effects on the growth of clonal tumor cells that differentially produce type IV collagen and laminin. Two separate clones derived from rat mammary carcinoma cells that produce high and low amounts of type IV collagen and laminin were injected into the flanks of nude mice. Tumors in animals receiving CHP treatment grew faster than tumors in control animals receiving saline, although statistically not significant. Furthermore, upon administration of CHP to these clones in culture, increased proliferation rates of both cell types were observed. These results show that CHP is not useful in preventing stromal development and growth of rat mammary tumor xenografts.

Most anti-inflammatory agents used in the treatment of joint diseases exert inhibitory effects on leukocyte infiltration. Methotrexate, a disease-modifying drug, and corticosteroids also inhibit leukocyte accumulation during inflammation. However, the mechanisms of action of these different compounds on leukocytes vary and in the case of non-steroidal anti-inflammatory drugs (NSAIDs) the mechanism(s) may be indirect. No current drug for inflammatory or degenerative joint disease has been proposed to act specifically by an inhibitory action on neutrophilic leukocytes. Oxaceprol is an amino acid derivative that has been used for several years for the treatment of osteoarthritis and rheumatoid arthritis, ameliorating pain and stiffness and showing good gastrointestinal safety, particularly in comparison with NSAIDs. Recent experimental studies have shown that oxaceprol does not inhibit the synthesis of prostaglandins in vitro, but markedly inhibits neutrophil infiltration into the joints of rats with adjuvant arthritis. These results support earlier screening data showing inhibition by oxaceprol of leukocyte infiltration into sites of acute inflammation. In studies on surgical ischemia reperfusion in hamsters in vivo, oxaceprol was an effective inhibitor of leukocyte adhesion and extravasation. It is proposed that oxaceprol represents a therapeutic agent for degenerative and inflammatory joint diseases, which acts predominantly by inhibiting leukocyte adhesion and migration.

Oxaceprol, an established drug for treatment of degenerative joint disease, has recently been shown in vitro to reduce leukocyte adhesion to cultured endothelial cells and leukocyte extravasation in vivo in an arthritis animal model. The aim of this study was to examine the effects of Oxaceprol on the microcirculation of striated skin muscle. In the dorsal skinfold chamber of the awake Syrian golden hamster I/R injury was induced by applying a 4-hour complete pressure ischemia. Prior to the ischemia, after 30 minutes, 2 hours and 24 hours of reperfusion the following parameters were assessed in a blinded study: macromolecular leakage, leukocyte rolling fraction, adherent leukocytes, and functional capillary density (FCD). Rhodamine 6G to stain leukocytes in-vivo and FITC Dextran (MW 150,000) was used as a plasma marker. 15 minutes prior to reperfusion the animals received either an i.v. bolus infusion of Oxaceprol (50mg/kg) or an equivalent volume of saline, which was followed by a 45-minute continuous infusion at the same dose. At the conclusion of the experiment samples were collected from the chamber tissue for histological quantification of leukocyte extravasation using an esterase stain. Oxaceprol treatment resulted in a significant decrease of postischemic leukocyte adherence after 0.5 h and 2h of reperfusion. The histological sections revealed a significant reduction in the number of extravasated leukocytes. There was a reduction of macromolecular leakage and treatment also resulted in a preservation of tissue perfusion, as was indicated by a significant increase in FCD in the treatment group compared to the ischemia group. In summary, Oxaceprol was able to protect the tissue from ischemia/reperfusion injury.

The present data indicate that treatment with Oxaceprol results in a significant protection of the microvasculature against I/R injury. The infusion of Oxaceprol during reperfusion leads to a significant reduction in the post-ischemic leukocyte adherence and extravasation as well as an improvement of the nutritive perfusion of the skin muscle. Thus, Oxaceprol treatment would appear to be a promising therapy for the reduction of I/R injury.

The proline analogue cis-4-hydroxy-L-proline (cHyp) inhibits collagen accumulation but diffuses out of tissues. To prolong the antifibrotic effect, we used a copolymer of cHyp attached to a backbone of poly(ethylene glycol) (PEG) and lysine. The copolymer was encapsulated in liposomes conjugated with PEG or in liposomes coated with the polysaccharide amylopectin to improve uptake by lungs after intravenous infusion. Amylopectin-liposomes had approximately 3-fold greater uptake in cultured endothelial cells compared with PEG-liposomes and greater lung retention 1 wk after infusion (5.2 +/- 0.8% versus 2.7 +/- 0.2%, p < 0.05). Sustained antifibrotic activity, assayed by growth inhibition of smooth muscle cells and fibroblasts over 4 d, was greater for amylopectin-liposomes/copolymer than PEG-liposomes/copolymer. Inhibition of collagen accumulation in pulmonary arteries of hypoxic (10% O2) rats was used to assess antifibrotic activity. Amylopectin-liposomes/copolymer attenuated increased right ventricular pressure by approximately 50% and completely prevented excess vascular collagen 1 wk after a single intravenous injection. The copolymer in liposomes was > 1,000-fold more effective by weight than unencapsulated monomeric cHyp. Thus, the copolymer, a potent, long-acting antifibrotic agent, totally prevented collagen accumulation for 1 wk in pulmonary arteries undergoing vascular remodeling when delivered in amylopectin-liposomes.

Purpose: Proliferative vitreoretinopathy (PVR) is characterized by the proliferation and migration of retinal pigment epithelial (RPE) and other cells into the vitreous cavity. The PVR membrane formation also is associated with collagen production by RPE. The authors examined the effect of a proline analog, cis-hydroxyproline (CHP), on proliferation, collagen synthesis, attachment, and migration of bovine RPE in vitro., Methods: The effect of CHP on cell proliferation was determined as a function of dosage and days in culture by counting the cell numbers on days 3, 6, and 9. Collagen synthesis was determined by trichloroacetic acid precipitation of the radiolabeled samples before and after bacterial collagenase digestion. The attachment assay involved type I collagen or fibronectin substrates or both (2.5 micrograms/well). For migration experiments, RPE cells were removed from a defined area of a confluent culture, and migration was quantitated by counting the number of cells migrating into the denuded area over 30 hours., Results: The addition of CHP inhibited RPE proliferation in both a dose- and a time-dependent manner; collagen synthesis, attachment, and migration also were inhibited by CHP in a dose-dependent manner. When the culture plates were coated with collagen, < 100 micrograms/ml of CHP had no effect on cell attachment. Higher doses of CHP resulted in mild inhibition of attachment on collagen-coated plates. Simultaneous addition of L-proline to the cultures resulted in blockade of these inhibitory effects on proliferation, collagen synthesis, attachment, and migration., Conclusions: The results show that RPE functions critical to the development of PVR are inhibited by CHP, suggesting the possibility that this drug may have potential clinical application.

Evidence from a variety of sources indicates that the extracellular matrix forms an important part of a feedback loop governing the migration, proliferation, and differentiation of the cells that produce it. In keeping with this, we showed previously that the extracellular matrix of a multipotential mesenchymal clonal cell line (ROB-C26) induced to differentiate into a more osteoblastic cell type by the addition of exogenous retinoic acid produces an extracellular matrix capable of osteoinductive activity in vivo and of stimulating alkaline phosphatase activity in vitro. Since type I collagen is the major structural component of this extracellular matrix, we sought to determine whether and to what extent this protein is responsible for the previously observed inductive/stimulatory activity. To this end, C26 cells are cultured on plastic, in the presence of retinoic acid, on a type I collagen film, or on an extracellular matrix from retinoic acid-treated C26 cells, and cell differentiation is assessed by measuring changes in the abundance of a number of osteoblast-related mRNAs. These determinations are made by RNAse protection assay after 3 or 6 days of incubation and include measurements of the RNAs for type I collagen, alkaline phosphatase, osteopontin, transforming growth factor alpha 1 and beta 2, and Vgr-1/BMP-6. In addition, C26 cells are incubated in the presence of retinoic acid and several established inhibitors of the synthesis or assembly of extracellular matrix components and the effects on induced alkaline phosphatase activity determined. Our data show that while the collagen substrate mimics some of the effects of retinoic acid and the extracellular matrix, it cannot reproduce all of them. Specifically, while the latter two culture conditions increase the abundance of all six mRNAs, type I collagen film increases the levels of only three of the six (collagen I, alkaline phosphatase, and osteopontin). Moreover, while type I collagen film produces an increase in alkaline phosphatase message, it falls to produce a similar change in alkaline phosphatase activity, an effect seen with both retinoic acid and extracellular matrix. However, interruption of collagen I synthesis by cis-4-hydroxy-L-proline blocks the increase in alkaline phosphatase activity associated with retinoic acid treatment. Thus, it appears likely that type I collagen is a necessary but, by itself, insufficient factor to elicit the comprehensive expression of the osteoblastic phenotype in immature mesenchymal cells.

A new series of 6-(hydroxyethyl)penems 2-substituted with amino acid-related side chains was synthesized. The nature of the amino acyl derivative proved to be crucial both from a synthetic point of view, as beta-lactam ring opening can compete with C-2 nucleophilic substitution, and for antibacterial activity. Primary amino acid amides emerged as the most suitable side chains for enhancing permeability through a Gram-negative outer membrane. In vitro activity of the new 2-[(aminoamido)methyl]penems 3a-u was influenced by the nature and position of the amide moiety, the ring size for cyclic amides, and the configuration of the amino acid. Compounds bearing amides derived from small N-methyl amino acids (such as 3a) or from cyclic amino acids (such as prolinamide 3p and 4-hydroxyprolinamide 3r) showed broad spectrum in vitro activity against both Gram-positive and Gram-negative microorganisms.

The central activity of hydroxyproline - the amino acid characteristic for collagen degradation products was studied. The levels of urinary and serum hydroxyproline are elevated in many disturbances, among them is a mental deficiency connected with hydroxyprolinemia. Previous studies showed that the mixture of collagen degradation products is biologically active and exerts and effect on central nervous system. In our study hydroxyproline significantly decreased the psychomotor activity of rats in Lat's test, enhanced amphetamine stereotypy in lower doses, and enhanced catalepsy in one of used doses. It prolonged the time of thiopental sleeping. The pentylenetetrazole seizures latency was not significantly prolonged and duration of seizures was shorter after injection (icv) of hydroxyproline. Our investigations show, that hydroxyproline is the active amino acid in the mixture of collagen degradation products and may be responsible for central activity of this mixture.

Synthetic approaches for the preparation of macromolecular conjugates of the antifibrotic agent cis-4-hydroxy-L-proline (cHyp) were explored, and the efficacy of the conjugates in inhibiting collagen accumulation was investigated in vitro and in vivo. In one approach, poly(PEG-Lys), an alternating copolymer of poly(ethylene glycol) (PEG) and lysine, was used as the carrier. To prepare pendent chain systems, cHyp was attached to poly(PEG-Lys) through an amide linkage [poly(PEG-Lys-cHyp amide)] or through an ester linkage [poly(PEG-Lys-cHyp ester)]. In an alternative approach, cHyp was incorporated into the backbone of a linear copolymer consisting of PEG, succinic acid, and cHyp units [poly(PEG-succinate-cHyp)]. Bioactivity in vitro was assessed by the ability of the cHyp conjugates to inhibit growth of cultured smooth muscle cells (SMC) and rat lung fibroblasts (RLF). Cell numbers were compared to control experiments in the presence of biologically inactive trans-4-hydroxy-L-proline (tHyp). After a 5 day period, the presence of 8 micrograms/mL of cHyp delivered by poly(PEG-Lys-cHyp amide) resulted in a 47% reduction in the number of SMC (p < 0.05), the presence of 36 micrograms/mL of cHyp delivered by poly(PEG-Lys-cHyp ester) resulted in a 38% reduction in the number of SMC (p < 0.05), while the presence of 118 micrograms/mL of cHyp delivered by poly(PEG-succinate-cHyp) resulted in a 31% reduction in the number of cells (p < 0.05). An identical trend was observed for the inhibition of RLF growth. In general, poly(PEG-Lys-cHyp amide) was most active, followed by poly(PEG-Lys-cHyp ester) and the backbone system, poly(PEG-succinate-cHyp). Specifically, poly(PEG-Lys-cHyp amide) was over 100-fold more active in inhibiting cell growth than free cHyp. Bioactivity in vivo was evaluated by measuring collagen accumulation in subcutaneously implanted poly(vinyl alcohol) sponges in rats. Among the tested conjugates, poly(PEG-Lys-cHyp amide) was most active, reducing collagen accumulation in the sponge by 33% after 14 days relative to controls (p < 0.05). This result indicates that the covalent attachment of cHyp to poly(PEG-Lys) carries may be a useful strategy for the local inhibition of collagen accumulation in tissues.

The extra-cellular matrix has been demonstrated to play an important role in the differentiation of a number of cell types in vitro. The purpose of this study was to establish the role of ECM collagen synthesis in regulating growth and differentiation of embryonic cardiocytes in vitro. We report that treatment of embryonic cardiocytes in vitro with two chemically distinct inhibitors of collagen synthesis, cis-hydroxy-L-proline and ethyl-3,4-dihydroxybenzoate, effectively inhibits collagen secretion. This results in disruption of myofibrillogenesis as seen by immunocytochemistry and electron microscopy, and absence of beating. The expression of muscle specific genes TroponinT and Myosin Heavy Chain are reduced, while the expression of the housekeeping gene glyceraldehyde phosphate dehydrogenase is uneffected. All of these effects are reversible. The structural effects are not prevented by growing the cells on various substrates, including denatured collagen, collagen type IV, laminin and Matrigel. Thus, inhibition of collagen secretion disrupts myofibrillogenesis and results in the alteration of expression of muscle-specific genes, suggesting that collagen synthesis plays an essential role in maintaining the differentiated phenotype of cardiocytes.

Mitochondrial response to the effect of a hydroxylase (PH) inhibitor was tested in marrow stromal cells during stimulation of osteoprogenitor cell (OPC) differentiation. The rationale for this was to explore pathways of regulatory interactions between procollagen synthesis and mitochondrial respiration that could be linked to the commitment of OPCs to mineralization. Stimulated OPCs exposed to the PH inhibitor cis-hydroxyproline (cis-HP), compared to the noninhibiting isomer trans-HP, for 11 days showed a dose-dependent decrease in cell proliferation, the surviving cells showed increased alkaline phosphatase activity. Trans-HP did not influence the cis-HP effect on ALP and on proliferation arrest. Short time exposures, 2-3 days, to cis-HP at different periods suggested that Days 0-3 and 3-5 were critical for the commitment to Day 21 mineralization of OPCs. On Days 0-3 cells were most sensitive to cis-HP, since on Day 11, 8 days after removal of cis-HP, they were too scarce to be counted by the staining method. However, the presence of 5.0 mM cis-HP in the cultures during Days 3-5 has induced on Day 21 close to 24-fold more mineralization/cell than controls, compared to the trans-HP effect, which was only close to 3-fold. The presence of cis-HP in the cultures on Days 0-3 has augmented the mitochondrial Day 3 retention of rhodamine 123 (Rho) in the stromal cells, relative to controls, compared to the presence of trans-HP. However, the presence of cis-HP during Days 3-5 or 3-6 resulted in lower Day 5 Rho retention, relative to controls, which was not significantly different from the retention that resulted from trans-HP. Since Rho retention is related to the final result of aerobic respiration level, these results are interpreted as a cis-HP inhibitory effect on procollagen peptidyl-proline hydroxylation, which may in turn release oxygen surpluses, to be available for mitochondrial consumption. The fall in Rho retention responses to cis-HP between Days 0-3 and 3-5 is suggesting either abrupt decrease in proline hydroxylation or poor mitochondrial sensitivity to oxygen in Day 3-5 cultures.

The pharmacological activity of glycine (Gly), proline (Pro) and hydroxyproline (Hyp)--the main amino acids in collagen degradation products--was studied in comparison to the activity of all mixture of DPTC (degradation products of trihelical collagen). The investigated amino acids decreased psychomotor activity of rats, enhanced apomorphine- and amphetamine-induced stereotypy. Pro and Hyp enhanced also catalepsy, but Gly decreased the haloperidol action. Each amino acid was given adequately to its contents in DPTC. Our results indicated that hydroxyproline revealed the action on the central nervous system (CNS) in each aspect most similar to the action of DPTC mixture.

Recruitment of keratocytes into injured corneal stroma, and secretion of proteins including collagen in the cells are essential for wound healing of the corneal stroma. We examined the effect of a proline analog, cis-hydroxyproline, on the adhesion, migration and growth of rabbit keratocytes in vitro. This agent decreased the plating efficiency, migration and growth of the keratocytes in a dose-dependent manner. Reduction in these cellular activities may reflect altered functions of pericellular proteins such as collagen. Further studies are needed to determine which specific protein is involved.

The present study examined the effects of cis-4-hydroxy-L-proline (CHP), a proline analog, on the androgen-induced growth of the lateral prostate of prepubertally castrated guinea pigs. Prepubertal male guinea pigs were castrated at the age of 3 weeks and allowed to recover completely before subjection to an experimental regime to alter the stromal collagen synthesis by CHP. The animals were kept on a special proline-deficient diet (PDD) for a week before the subcutaneous injection of CHP (200 mg/kg/day) for 3 days, followed by a combined injection of CHP and dihydrotestosterone (DHT) (10 mg/kg/day) for 10 days. Control animals were injected with saline and DHT only. At the end of the experiment, the lateral prostate was removed and examined by 1) conventional transmission electron microscopy (TEM), 2) staining of proteoglycans (PGs) by Cuprolinic Blue (CB) using the critical electrolyte concentration (CEC) method, 3) carbohydrate and lectin histochemistry, and 4) electron microscopy (EM) lectin-gold labelling. The results showed that the wet weight of prostate from CHP-treated animals was significantly lower than the control and recovery groups. The epithelium was low columnar with an obvious increase in intercellular spaces and number of basal cells. The glandular cells showed little secretory activity with a decrease in number of granular endoplasmic reticulum (GER) profiles, secretory granules, and a small Golgi apparatus. The stroma was composed of stromal cells separated by large intercellular spaces with very sparse collagen fibrils, and a decrease in stromal PGs especially those PGs normally associated with collagen fibrils. CHP treatment also caused perturbation and disorganization in the epithelial basement membrane. The results suggested that stromal collagen is essential in mediating the response of glandular cells to DHT stimulation. Defective stromal collagens hamper the responsiveness of prostatic gland to androgen.

cis-4-Hydroxy-L-proline (cis-OH-Pro) and ethyl-3,4-dihydroxybenzoate (EDHB), two distinct inhibitors of collagen synthesis, prevented myogenesis in C2C12 mouse skeletal muscle cells. Both inhibitors blocked myotube formation and the expression of sarcomeric myosin heavy chain. Northern blot analysis showed that cis-OH-Pro- and EDHB-treated C2C12 muscle cells did not express the myogenic regulatory genes, MyoD1 and myogenin, but continued to express non-muscle isoforms of actin (beta and gamma) and alpha-tropomyosin. 10TFL2-3B cells, a C3H10T1/2 cell line permanently transfected with myogenin cDNA, constitutively expressed exogenous myogenin in the presence of cis-OH-Pro but failed to activate endogenous myogenin and to undergo myogenesis. These results demonstrate that commitment to terminal differentiation and activation of myogenic regulatory genes requires active synthesis of the extracellular matrix component collagen.

We investigated antigenic changes of murine glomerular basement membrane (GBM) collagen type IV after oral feedings of 3-cis- or 4-cis-hydroxyproline. As shown by thin layer chromatography and gas chromatographic-mass spectrometric studies, 3-cis- and 4-cis-hydroxyproline were incorporated into the GBM collagen instead of the normal trans isomer and led to a considerable reduction of the collagen antibody reactivity on indirect immunofluorescence, passive hemagglutination and radioimmunoassay when compared to GBM collagen of control animals. We speculate that antigen modification due to the incorporation of stereoisomers could serve as a model for basement membrane immunopathology.