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A key approach for designing bioinspired machines is to transfer concepts from nature to man-made structures by integrating biomolecules into artificial mechanical systems. This strategy allows the conversion of molecular information into macroscopic action. Here, we describe the design and dynamic behaviour of hybrid bioelectrochemical swimmers that move spontaneously at the air-water interface. Their motion is governed by the diastereomeric interactions between immobilized enantiopure oligomers and the enantiomers of a chiral probe molecule present in solution. These dynamic bipolar systems are able to convert chiral information present at the molecular level into enantiospecific macroscopic trajectories. Depending on the enantiomer in solution, the swimmers will move clockwise or anticlockwise; the concept can also be used for the direct visualization of the degree of enantiomeric excess by analysing the curvature of the trajectories. Deciphering in such a straightforward way the enantiomeric ratio could be useful for biomedical applications, for the read-out of food quality or as a more general analogue of polarimetric measurements., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

The pancreatic β cells can synthesize dopamine by taking L-dihydroxyphenylalanine, but whether pancreatic acinar cells synthesize dopamine has not been confirmed. By means of immunofluorescence, the tyrosine hydroxylase -immunoreactivity and aromatic amino acid decarboxylase (AADC)- immunoreactivity were respectively observed in pancreatic acinar cells and islet β cells. Treatment with L-dihydroxyphenylalanine, not tyrosine, caused the production of dopamine in the incubation of INS-1 cells (rat islet β cell line) and primary isolated islets, which was blocked by AADC inhibitor NSD-1015. However, only L-dihydroxyphenylalanine, but not dopamine, was detected when AR42J cells (rat pancreatic acinar cell line) were treated with tyrosine, which was blocked by tyrosine hydroxylase inhibitor AMPT. Dopamine was detected in the coculture of INS-1 cells with AR42J cells after treatment with tyrosine. In an in vivo study, pancreatic juice contained high levels of L-dihydroxyphenylalanine and dopamine. Both L-dihydroxyphenylalanine and dopamine accompanied with pancreatic enzymes and insulin in the pancreatic juice were all significantly increased after intraperitoneal injection of bethanechol chloride and their increases were all blocked by atropine. Inhibiting TH with AMPT blocked bethanechol chloride-induced increases in L-dihydroxyphenylalanine and dopamine, while inhibiting AADC with NSD-1015 only blocked the dopamine increase. Bilateral subdiaphragmatic vagotomy of rats leads to significant decreases of L-dihydroxyphenylalanine and dopamine in pancreatic juice. These results suggested that pancreatic acinar cells could utilize tyrosine to synthesize L-dihydroxyphenylalanine, not dopamine. Islet β cells only used L-dihydroxyphenylalanine, not tyrosine, to synthesize dopamine. Both L-dihydroxyphenylalanine and dopamine were respectively released into the pancreatic duct, which was regulated by the vagal cholinergic pathway. The present study provides important evidences for the source of L-dihydroxyphenylalanine and dopamine in the pancreas.

Tyrosinase is the key enzyme for the metabolism of tyrosine and inherently comprises both monophenolase activity and diphenolase activity. A real-time fluorometric assay method was established to exclusively monitor the monophenolase activity by eliminating interference from diphenolase reactions through a combination of borate and hydroxylamine. Synthetic matrices comprised of tyrosine and DOPA (L-3,4-dihydroxyphenylalanine) preincubated with tyrosinase with the consistent sum concentration of 70 μM to mimic the monophenolase reaction mixture in borate buffer according to law of mass conservation. A matrix-matched calibration curve for determination of tyrosine was established using the synthetic matrices as standard sample to eliminate spectral interference from DOPA. The limit of detection (LOD) for tyrosine was 0.61 μM. The time course for consumption of tyrosine was established to measure the initial velocity through real-time reading out the tyrosine fluorescence intensity of the reaction mixture in a cuvette in situ. The assay worked in the monophenolase activity range from 0.2839 to 1.7308 U mL -1 with LOD of 0.0851 U mL -1 . The proposal sensing system successfully afforded a prospective potential for application in enzyme kinetics and screening of inhibitor. Graphical abstract.

This work demonstrates the use of a fluorescent probe to screen protein conformational changes in mixtures of monoclonal antibodies and determine the region of where such changes may originate through a footprinting mass spectrometry approach. The oxidative stress of mixtures of two different immunoglobulins (IgG1, IgG2, or IgG4) performed in the presence of 2,2'-azobis(2-amidinopropane dihydrochloride) results in sequence-specific tyrosine oxidation reactions depending on the time of incubation of the IgG molecules and the nature of the excipients present in the formulation. The combination of a fluorescence assay, based on the detection of 3,4-dihydroxyphenylalanine (DOPA) and mass spectrometry analyses, permits the identification of protein conformation changes. In a mixture of IgG2 and IgG4, a destabilization of IgG4 in the presence of IgG2 is observed. The destabilized region involves the Fab region of IgG4 between Tyr63 and Tyr81 and potentially multiple regions of IgG2.

Numerous chiral drugs have been developed for neurological diseases, but chiral drug safety and pharmacological research still faces great challenges in enantioselectivity and sensitivity of chiral analysis. A rapid, stable and high-efficiency gold nanodendrite-based electrochemical sensing method was proposed as a versatile differential potential ratiometric strategy for highly selective chiral recognition of dual 3,4-dihydroxyphenylalanine (DOPA) enantiomers. Using simple electrochemical deposition, the gold nanodendrite (AuND) membrane was steadily modified on the glassy carbon electrode (GCE) with high specific chiral identifiability and robust signal amplifiability. Based on a sequence of systematic optimization, an apparent potential difference (PD) was detected up to 108 mV from oxidation peaks of DOPA enantiomers. Furthermore, according to a regulatable concentration range from 10 μM to 100 μM, different proportions of L-/d-DOPA had a good linear relationship with corresponding peak potentials (Eps) in the racemic mixture. Superior to traditional enantiorecognition of single chiral drug, both DOPA enantiomers enabled to be simultaneously distinguished with high repeatability, selectivity, and anti-interference ability on the AuND/GCE. This unique semi-quantitative AuND-based potential sensing strategy was proposed to efficiently quantify the proportion of L-/d-DOPA for chiral drug recognition, emerging positive potential for numerous applications in pharmacology and clinical diagnosis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Using a surface forces apparatus and an atomic force microscope, we characterized the adhesive properties of adsorbed layers of two recombinant variants of Perna viridis foot protein 5 (PVFP-5), the main surface-binding protein in the adhesive plaque of the Asian green mussel. In one variant, all tyrosine residues were modified into 3,4-dihydroxy-l-phenylalanine (DOPA) during expression using a residue-specific incorporation strategy. DOPA is a key molecular moiety underlying underwater mussel adhesion. In the other variant, all tyrosine residues were preserved. The layer was adsorbed on a mica substrate and pressed against an uncoated surface. While DOPA produced a stronger adhesion than tyrosine in contact with the nanoscopic Si 3 N 4 probe of the atomic force microscope, the two variants produced comparable adhesion on the curved macroscopic mica surfaces of the surface forces apparatus. These findings show that the presence of DOPA is not a sufficient condition to generate strong underwater adhesion. Surface chemistry and contact geometry affect the strength and abundance of protein-surface bonds created during adsorption and surface contact. Importantly, the adsorbed protein layer has a random and dynamic polymer-network structure that should be optimized to transmit the tensile stress generated during surface separation to DOPA surface bonds rather than other weaker bonds.

Introduction: Medullary carcinoma accounts for 1-2% of all thyroid malignancies. 13-20% of patients present with distant metastasis, with 45% of the cases affecting the liver., Clinical Case: A 50-year-old woman, diagnosed with medullary thyroid carcinoma, was treated with total thyroidectomy and a modified neck dissection in 1999. Two lymph node recurrences in the neck were treated with surgical resection; during surveillance, she developed elevated calcitonin levels, the recurrence site was identified with 18 F-DOPA PET/CT in the liver. Metabolic activity was not associated with a visible lesion in CT, MRI nor ultrasound. Radioguided surgery with 18 F-DOPA allowed an anatomic resection of segments IVb and V., Discussion: In patients with medullary carcinoma and elevated calcitonin during surveillance, 18 F-DOPA PET/CT is an option to evaluate the site of recurrence. Radioguided resection was feasible in this patient, whose hepatic recurrence was not visible with any other imaging method., Conclusion: Radioguided hepatic resection with 18 F-DOPA in metastatic medullary thyroid carcinoma is feasible when the recurrence site is not anatomically identified by any other imaging studies., (Copyright © 2017. Publicado por Elsevier España, S.L.U.)

According to one hypothesis, Parkinson's disease pathogenesis is largely caused by dopamine catabolism that is catalyzed on mitochondrial membranes by monoamine oxidase. Reactive oxygen species are formed as a byproduct of these reactions, which can lead to mitochondrial damage followed by cell degeneration and death. In this study, we investigated the effects of administration of the mitochondrial antioxidant SkQ1 on biochemical, immunohistochemical, and behavioral parameters in a Parkinson-like condition caused by protoxin MPTP injections in C57BL/6 mice. SkQ1 administration increased dopamine quantity and decreased signs of sensory-motor deficiency as well as destruction of dopaminergic neurons in the substantia nigra and ventral tegmental area in mice with the Parkinson-like condition.

Cocaine is a recreational drug of abuse that binds to the dopamine transporter, preventing reuptake of dopamine into pre-synaptic terminals. The increased presence of synaptic dopamine results in stimulation of both pre- and post-synaptic dopamine receptors, considered an important mechanism by which cocaine elicits its reinforcing properties. However, the effects of acute cocaine administration on pre-synaptic dopamine function remain unclear. Non-invasive imaging techniques such as positron emission tomography have revealed impaired pre-synaptic dopamine function in chronic cocaine users. Similar impairments have been seen in animal studies, with microdialysis experiments indicating decreased basal dopamine release. Here we use micro positron emission tomography imaging techniques in mice to measure dopamine synthesis capacity and determine the effect of acute cocaine administration of pre-synaptic dopamine function. We show that a dose of 20 mg/kg cocaine is sufficient to elicit hyperlocomotor activity, peaking 15-20 min post treatment (p < 0.001). However, dopamine synthesis capacity in the striatum was not significantly altered by acute cocaine treatment (KiCer: 0.0097 per min vs. 0.0112 per min in vehicle controls, p > 0.05). Furthermore, expression levels of two key enzymes related to dopamine synthesis, tyrosine hydroxylase and aromatic l-amino acid decarboxylase, within the striatum of scanned mice were not significantly affected by acute cocaine pre-treatment (p > 0.05). Our findings suggest that while the regulation of dopamine synthesis and release in the striatum have been shown to change with chronic cocaine use, leading to a reduced basal tone, these adaptations to pre-synaptic dopaminergic neurons are not initiated following a single exposure to the drug., (© 2017 International Society for Neurochemistry.)

Discriminative sensing of chiral species with a convenient and robust system is a challenge in chemistry, pharmaceutics and particularly in biomedical science. Advanced nanohybrid materials for discrimination of these biologically active molecules can be developed by combination of individual obvious advantages of different molecular scaffolds. Herein, we report on the comparison of the performance of cyclodextrin functionalized graphene derivatives (x-CD/rGO, x: α-, β-, γ-) for discrimination of DOPA enantiomers. Within this respect, electrochemical measurements were conducted and the experimental results were compared to molecular docking method. Thanks to cavity size of γ-CD and the unique properties of graphene, rGO/γ-CD nanohybrid is capable of selective recognition of DOPA enantiomers. Limit of detection (LOD) value and sensitivity were determined as 15.9 μM and 0.2525 μA μM -1 for D-DOPA, and 14.9 μM and 0.6894 μA μM -1 for L-DOPA., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Robust adhesion to wet, salt-encrusted, corroded and slimy surfaces has been an essential adaptation in the life histories of sessile marine organisms for hundreds of millions of years, but it remains a major impasse for technology. Mussel adhesion has served as one of many model systems providing a fundamental understanding of what is required for attachment to wet surfaces. Most polymer engineers have focused on the use of 3,4-dihydroxyphenyl-l-alanine (Dopa), a peculiar but abundant catecholic amino acid in mussel adhesive proteins. The premise of this Review is that although Dopa does have the potential for diverse cohesive and adhesive interactions, these will be difficult to achieve in synthetic homologs without a deeper knowledge of mussel biology; that is, how, at different length and time scales, mussels regulate the reactivity of their adhesive proteins. To deposit adhesive proteins onto target surfaces, the mussel foot creates an insulated reaction chamber with extreme reaction conditions such as low pH, low ionic strength and high reducing poise. These conditions enable adhesive proteins to undergo controlled fluid-fluid phase separation, surface adsorption and spreading, microstructure formation and, finally, solidification., (© 2017. Published by The Company of Biologists Ltd.)

Background: Tranexamic acid has an inhibitory action on ultraviolet (UV) B-induced melanocyte activation. This study examined the sex differences in the inhibitory action of tranexamic acid on UVB-induced melanocyte activation., Methods: We irradiated the eye and ear of male and female mice with UVB at a dose of 1.0 kJ/m(2) using a 20SE sunlamp. We orally administered tranexamic acid (750 mg/kg/day) at 30 min before UVB exposure., Results: Tranexamic acid inhibited the UVB-induced epidermal melanocyte activation, and the effect was more remarkable under UVB eye irradiation than under UVB ear irradiation. Furthermore, the melanocyte activity suppression effect was stronger in female mice than in male mice. Following the administration of tranexamic acid, the female displayed increased blood levels of β-endorphin and μ-opioid receptor and estradiol receptor β expression in comparison with the male. Furthermore, the effect of melanocyte activity suppression in the female mice was decreased by the administration of tamoxifen (antagonist of estrogen receptor) or naltrexone (antagonist of μ-opioid receptor)., Conclusions: These results suggest that the suppression by tranexamic acid of the UVB-induced melanocyte activation (UVB sensitivity) is stronger in female mice than in male mice and that female hormones and β-endorphin play an important role in this sex difference., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Neuroblastoma (NB) is a heterogeneous childhood cancer that requires multiple imaging modalities for accurate staging and surveillances. This study aims to investigate the utility of positron emission tomography (PET) with 18F-fluorodeoxyglucose (FDG) and 18F-fluoro-dihydroxyphenylalanine (FDOPA) in determining the prognosis of NB. During 2007-2014, forty-two NB patients (male:female, 28:14; median age, 2.0 years) undergoing paired FDG and FDOPA PET scans at diagnosis were evaluated for the maximum standardized uptake value (SUV(max)) of FDG or FDOPA by the primary tumor. Patients with older age, advanced stages, or MYCN amplification showed higher FDG and lower FDOPA SUV(max) (all P < 0.02). Receiver operating characteristics analysis identified FDG SUV(max) ≥ 3.31 and FDOPA SUV(max) < 4.12 as an ultra-high-risk feature (PET-UHR) that distinguished the most unfavorable genomic types, i.e. segmental chromosomal alterations and/or MYCN amplification, at a sensitivity of 81.3% (54.4%-96.0%) and a specificity of 93.3% (68.1%-99.8%). Considering with age, stage, MYCN status, and anatomical image-defined risk factor, PET-UHR was an independent predictor of inferior event-free survival (multivariate hazard ratio, 4.9 [1.9-30.1]; P = 0.012). Meanwhile, the ratio between FDG and FDOPA SUV(max) (G:D) correlated positively with HK2 (Spearman's ρ = 0.86, P < 0.0001) and negatively with DDC (ρ = -0.58, P = 0.02) gene expression levels, which might suggest higher glycolytic activity and less catecholaminergic differentiation in NB tumors taking up higher FDG and lower FDOPA. In conclusion, the intensity of FDG and FDOPA uptake on diagnostic PET scans may predict the tumor behavior and complement the current risk stratification systems of NB.

Benzylisoquinoline alkaloids (BIAs) are a diverse family of plant-specialized metabolites that include the pharmaceuticals codeine and morphine and their derivatives. Microbial synthesis of BIAs holds promise as an alternative to traditional crop-based manufacturing. Here we demonstrate the production of the key BIA intermediate (S)-reticuline from glucose in Saccharomyces cerevisiae. To aid in this effort, we developed an enzyme-coupled biosensor for the upstream intermediate L-3,4-dihydroxyphenylalanine (L-DOPA). Using this sensor, we identified an active tyrosine hydroxylase and improved its L-DOPA yields by 2.8-fold via PCR mutagenesis. Coexpression of DOPA decarboxylase enabled what is to our knowledge the first demonstration of dopamine production from glucose in yeast, with a 7.4-fold improvement in titer obtained for our best mutant enzyme. We extended this pathway to fully reconstitute the seven-enzyme pathway from L-tyrosine to (S)-reticuline. Future work to improve titers and connect these steps with downstream pathway branches, already demonstrated in S. cerevisiae, will enable low-cost production of many high-value BIAs.

We demonstrate, for the first time, an electrochemical sensor that provides antipodal signals upon application of square wave voltammetry (SWV), for enantioselective recognition of 3,4-dihydroxyphenylalanine based on chiral single-walled carbon nanotubes (SWCNTs) in the presence of sulphuric acid. Interestingly, the enantioselectivity was not observed using the common method of cyclic voltammetry (CV) but the SWV peak currents of enantiomers were found to be quite different and hence the enantiomers could be successfully recognized. Moreover, the antipodal signals provided by two SWV scan modes offer the possibility for results to be confirmed mutually, showing a great practical value and analytical application prospects.

Many marine invertebrates utilize biomacromolecules as building blocks to form their load-bearing tissues. These polymeric tissues are appealing for their unusual physical and mechanical properties, including high hardness and stiffness, toughness and low density. Here, a marine hydroid perisarc of Aglaophenia latirostris was investigated to understand how nature designs a stiff, tough and lightweight sheathing structure. Chitin, protein and a melanin-like pigment, were found to represent 10, 17 and 60 wt.% of the perisarc, respectively. Interestingly, similar to the adhesive and coating of marine mussel byssus, a DOPA (3,4-dihydroxyphenylalanine) containing protein and iron were detected in the perisarc. Resonance Raman microprobe analysis of perisarc indicates the presence of catechol-iron(III) complexes in situ, but it remains to be determined whether the DOPA-iron(III) interaction plays a cohesive role in holding the protein, chitin and melanin networks together., (Copyright © 2013 Acta Materialia Inc. All rights reserved.)

L-3,4-dihydroxyphenylalanine (L-DOPA) is a well-recognized therapeutic compound to Parkinson's disease. Tyrosine is a precursor for the biosynthesis of L-DOPA, both of which are widely found in traditional medicinal material, Mucuna pruriens. In this paper, we described a validated novel analytical method based on microchip capillary electrophoresis with pulsed electrochemical detection for the simultaneous measurement of L-DOPA and tyrosine in M. pruriens. This protocol adopted end-channel amperometric detection using platinum disk electrode on a homemade glass/polydimethylsiloxane electrophoresis microchip. The background buffer consisted of 10 mM borate (pH 9.5) and 0.02 mM cetyltrimethylammonium bromide, which can produce an effective resolution for the two analytes. In the optimal condition, sufficient electrophoretic separation and sensitive detection for the target analytes can be realized within 60 s. Both tyrosine and L-DOPA yielded linear response in the concentration range of 5.0-400 μM (R(2) > 0.99), and the LOD were 0.79 and 1.1 μM, respectively. The accuracy and precision of the established method were favorable. The present method shows several merits such as facile apparatus, high speed, low cost and minimal pollution, and provides a means for the pharmacologically active ingredients assay in M. pruriens., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We developed a simple and rapid liquid chromatography/mass spectrometry (LC/MS) method for the enantiomeric determination of DOPA in dietary supplements containing Mucuna pruriens. L- and D-DOPA were ultrasonically extracted with 1% formic acid aqueous solution. The isolated extracts were analyzed by LC/MS using a Crownpak CR (-) column at 30℃. The mass spectrometer was operated in the positive mode of electrospray ionization, and the mobile phase was aqueous formic acid (pH 2.0). L-DOPA-ring-d3 was used as an internal standard. The method was validated for a dietary supplement spiked with L- and D-DOPA at 50 and 500 μg/g, respectively, and the recoveries of the DOPA enantiomers were between 97.5% and 101.3%. Relative standard deviation values of repeatability and intermediate precision were less than 7%. The method was applied to 14 dietary supplements. L-DOPA was detected in these supplements in the range of 0.88-12.8 mg/unit. D-DOPA was not detected.

Catecholamines, such as dopamine and L-dihydroxyphenylalanine (L-DOPA), are associated with different physiological functions and diseases. In our recent studies, a novel water-soluble boronic acid compound 3c was identified as a selective fluorescent sensor for L-DOPA. This compound not only has the ability to interact with dopamine and catechol, but also has no fluorescence intensity change for L-DOPA precursors in vivo, such as L-tyrosine.

A fully automated chiral capillary electrophoresis-tandem mass spectrometry (CE-MS/MS) method was developed for enantiomeric quantification of 3,4-dihydroxyphenylalanine (DOPA) and its precursors, phenylalanine (Phe) and tyrosine (Tyr). To avoid MS source contamination, a negatively charged chiral selector, sulfated β-cyclodextrin (sulfated β-CD), that migrated away from the detector was used in combination with the partial filling technique. The six stereoisomers were simultaneously quantified in less than 12 min. Detection limits were 0.48 and 0.51 μM for l- and d-DOPA enantiomers, respectively. Assay reproducibility values (relative standard deviations [RSDs], n=6) were 4.43, 3.15, 4.91, 5.16, 3.96, and 3.25% for l- and d-DOPA, l- and d-Tyr, and l- and d-Phe at 10 μM, respectively. Thanks to the high enantioseparation efficiency, detection of trace d-DOPA in l-/d-DOPA mixtures could be achieved. The assay was employed to study the metabolism of DOPA, a well-known therapeutic drug for treating Parkinson's disease. It was found that l-DOPA was metabolized effectively in PC-12 cells. Approximately 88% of l-DOPA disappeared after incubation at a cell density of 2×10(6)cells/ml for 3 h. However, d-DOPA coexisting with l-DOPA in the incubation solution remained intact. The enantiospecific metabolism of DOPA in this neuronal model was demonstrated., (Copyright © 2011 Elsevier Inc. All rights reserved.)

This work describes the reaction mechanism for chemical modification of tyrosinase by Woodward's Reagent K and its covalent attachment to a glassy carbon electrode. The spectrophotometric studies revealed that the modification does not cause a significant structural change to tyrosinase. The direct electrochemistry of modified enzyme was achieved after immobilization on an oxidatively activated glassy carbon electrode. The enzyme film exhibited a pair of well-defined quasi-revesible voltammetric peaks corresponding to the Cu (II)/Cu (I) redox couple located in the active site of tyrosinase. The formal potential of immobilized enzyme was measured to be 90mV (vs. Ag/AgCl) in phosphate buffer solution at pH 7.0. The charge-transfer coefficient and apparent heterogeneous electron transfer rate constant were estimated to be 0.5 and 0.9±0.06s(-1), respectively. Finally, the electrochemical behavior of the immobilized enzyme in the presence of caffeic acid and L-3,4-dihydroxyphenylalanine as substrates was investigated. The amperometric study of biosensor toward L-3,4-dihydroxyphenylalanine resulted a linear response in the concentration range from 1.66×10(-6) to 8.5×10(-5)M with detection limit of 9.0×10(-5)M and sensitivity of 135mAμM(-1)cm(-2)., (Copyright © 2011 Elsevier B.V. All rights reserved.)

A malfunctioning heater caused a severe carbon monoxide (CO) intoxication leading to unconsciousness and predominantly right-sided extrapyramidal syndrome in a 29-year-old man. Follow-up included thorough clinical monitoring, and brain MRI and PET studies. Nine days after the poisoning, brain MRI showed symmetrical necrosis in the globus pallidi, but no abnormality was found in the substantia nigra. In addition, white matter periventricular lesions were seen. In a control scan 14 months later the white matter changes had subsided but small necrotic lesions were still noted bilaterally in the globus pallidi. A 6-[(18)F]fluoro-L-dopa PET examination performed 5 weeks after the intoxication revealed impaired presynaptic dopaminergic function in the left putamen whereas in the right putamen the dopaminergic activity was within normal limits. [(11)C] raclopride PET imaging 4 months after the poisoning showed no abnormality in postsynaptic D2 binding in the striatum. Clinically, the parkinsonian symptoms resolved 1.5 years after the poisoning. The final outcome of the recovery was excellent, and the patient returned to work. This is the first case reported where unilateral presynaptic, dopaminergic hypofunction in putamen could be confirmed with fluoro-l-dopa PET imaging on a patient with extrapyramidal syndrome caused by CO poisoning. Our results emphasize that CO intoxication can lead to striatal dopaminergic hypofunction, and that PET is a sensitive tool in evaluating extrapyramidal system after sudden neurotoxic insult., ((c) 2010 Elsevier Inc. All rights reserved.)

Growth dynamics of the synthesis of monoamine neuromediators serotonin, norepinephrine, and dopamine in Escherichia coli K-12 was investigated for the first time using high performance liquid chromatography with electrodetection. Maximum (micromolar) concentrations of these compounds were detected in E. coli cells during the early growth phases; their intracellular content decreases after the transition to late growth phases. E. coli biomass contains the substances DOPA and 5-hydroxytryptamine that serve in animal cells as neuromediator precursors and the products of their oxidative deamination. Presumably, the biosynthesis and degradation of monoamine neuromediators in bacterial cells involves enzyme systems analogous to those typical of animals. The culture fluid of E. coli contains micromolar concentrations of DOPA and nanomolar of serotonin, dopamine, and norepinephrine during the late growth phase. These concentrations are sufficient for animal/human receptors to bind them. This article deals with the potential biotechnological applications of the data obtained.

A chiral microemulsion electrokinetic chromatography method has been developed for the enantiomeric separation of 3,4-dihydroxyphenylalanine (dopa), its precursors phenylalanine and tyrosine, and the structurally related substance methyldopa. The separations were achieved using an oil-in-water microemulsion, which consisted of the oil-compound ethyl acetate, the surfactant sodium dodecylsulfate (SDS), the co-surfactant 1-butanol, the organic modifier propan-2-ol and 20mM phosphate buffer pH 2.5 or 2.0 as aqueous phase. For enantioseparation sulfated beta-cyclodextrin was added. The resolution of each racemate was optimized by varying the concentration of the buffer and all components of the microemulsion. Enantioseparation could be achieved for dl-dopa, dl-phenylalanine and dl-tyrosine within 13 min with a resolution of 4.3, 3.1 and 3.3, respectively, and for methyldopa in 17 min (Rs: 1.4). The established methods allowed the detection of dopa, phenylalanine, tyrosine and methyldopa with a limit at 0.5, 1.0, 0.2 and 2.0 microg/ml.

Objectives: Plasma neopterin is a clinical marker of inflammation. Interferon-gamma triggers 7,8-dihydroneopterin and its oxidation product, neopterin, to be released from macrophages. 7,8-dihydroneopterin is a potent antioxidant which can protect macrophages from oxidative damage in vitro. This study examined whether 7,8-dihydroneopterin/neopterin levels reach sufficient concentrations in human pus to provide antioxidant activity and be the source of plasma neopterin., Design and Methods: Pus was removed by needle aspiration from 19 patients and examined for total neopterin, protein-bound DOPA, dityrosine, alpha-tocopherol, lipid oxidation and protein carbonyls., Results: Total neopterin was detected between 50 nM and 1.2 microM, with an average concentration of 0.51 microM. Significant quantities of oxidized proteins and lipids were detected. alpha-Tocopherol concentrations positively correlate with total neopterin levels., Conclusions: Total neopterin levels found in the pus was up to 100 times higher than that reported in plasma, suggesting plasma neopterin originates from inflammatory sites. The concentration of total neopterin suggests that 7,8-dihydroneopterin could act as an antioxidant during inflammation.

A simple and rapid qualitative liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed and validated for screening cyclo-dopa and diketopiperazine alkaloids in crude extracts of Portulaca oleracea L. at sub-ppm levels. An electrospray ionization orbitrap mass spectrometer, which provides accurate full scan MS and MS/MS data, was used in this study. After simple extraction with ethanol and purification by AB-8 resin, the extracts were subjected to LC/MS/MS analysis. A high mass tolerance (10 ppm) was used in the initial screening to filter the full scan MS data. The cyclo-dopa and diketopiperazine alkaloid standards gave limits of detection (LODs) at or below 5 ng/mL. The results also indicated that the method had an acceptable precision for day-to-day use in the identification of compounds. The alkaloids could be identified based on their MS/MS data, elemental compositions, and retention behavior. This system was used to assay trace amounts of cyclo-dopa and diketopiperazine alkaloids in crude extracts of Portulaca oleracea L., leading to the identification of 5/2 confirmed/unconfirmed cyclo-dopa and 7/6 confirmed/unconfirmed diketopiperazine alkaloids, respectively. The screening method considerably reduces the time and cost involved in the identification of cyclo-dopa and diketopiperazine alkaloids in Portulaca oleracea L., as well as being a simple and convenient approach to the identification of other structural families of natural products., (Copyright (c) 2008 John Wiley & Sons, Ltd.)

A fixed ligand (FL) version of the kinetic method was applied to rapid, simple, and accurate chiral analysis of DOPA, which is an important drug used for treatment of Parkinson's disease. Singly charged clusters containing the transition metal ion Cu(II), pyridyl ligands which serve as a fixed ligand, some amino acid as a reference, and the analyte DOPA were generated by electrospray ionization. The cluster ion of interest was mass-selected, and the kinetics of its competitive unimolecular dissociations was investigated in an ion trap mass spectrometer. The chiral selectivity (R(chiral)), the ratio of the two fragment ion abundances when the cluster contains one pure enantiomer of the analyte expressed relative to that for the other enantiomer, varies with fixed ligands, references, and transition metals. Chiral discrimination was optimized in 1,10-phenanthroline as a FL, L-Phe and L-Pro as a reference, and Cu(II) as a central metal ion. Quantitative determinations of the enantiomeric composition of DOPA were achieved using two-point calibration curves. The linear relationship between the logarithm of the fragment ion abundance ratio (ln R) and enantiomeric compositions (ee%) of the DOPA allows the determination of the chiral purity of enantiomeric mixtures., ((c) 2008 John Wiley & Sons, Ltd.)

The beak of the Humboldt squid Dosidicus gigas represents one of the hardest and stiffest wholly organic materials known. As it is deeply embedded within the soft buccal envelope, the manner in which impact forces are transmitted between beak and envelope is a matter of considerable scientific interest. Here, we show that the hydrated beak exhibits a large stiffness gradient, spanning two orders of magnitude from the tip to the base. This gradient is correlated with a chemical gradient involving mixtures of chitin, water, and His-rich proteins that contain 3,4-dihydroxyphenyl-L-alanine (dopa) and undergo extensive stabilization by histidyl-dopa cross-link formation. These findings may serve as a foundation for identifying design principles for attaching mechanically mismatched materials in engineering and biological applications.

The electrochemical behavior of redox centers in the active site of amine oxidases from lentil seedlings and Euphorbia characias latex was investigated using a mercury film electrode. Tyrosine-derived 6-hydroxydopa quinone (TPQ) and copper ions in the active site are redox centers of these amine oxidases. The enzymes undergo two reduction processes at negative potentials related to the reduction of the TPQ cofactor to the corresponding hydroquinones and the reduction of copper ions, (Cu(II)-->Cu(I)). Copper depleted enzymes, prepared by reduction with dithionite followed by dialysis against cyanide, undergo only one reduction process. Nyquist diagrams, recorded at potentials corresponding to the reduction of cofactors as dc-offset, represent charge transfer impedance followed by a Warburg-type line at low frequencies, indicating the occurrence of a diffusion controlled process in the rate-limiting step of the reduction process.

Melanosomes are unique membrane-bound organelles specialized for the synthesis and distribution of melanin. Mechanisms involved in the trafficking of proteins to melanosomes and in the transport of mature pigmented melanosomes to the dendrites of melanocytic cells are being characterized, but details about those processes during early stages of melanosome maturation are not well understood. Early melanosomes must remain in the perinuclear area until critical components are assembled. In this study, we characterized the processing of two distinct melanosomal proteins, tyrosinase (TYR) and Pmel17, to elucidate protein processing in early or late steps of the secretory pathway, respectively, and to determine mechanisms underlying the subcellular localization and transport of early melanosomes. We used immunological, biochemical, and molecular approaches to demonstrate that the movement of early melanosomes in the perinuclear area depends primarily on microtubules but not on actin filaments. In contrast, the trafficking of TYR and Pmel17 depends on cytoplasmic dynein and its interaction with the spectrin/ankyrin system, which is involved with the sorting of cargo from the plasma membrane. These results provide important clues toward understanding the processes involved with early events in melanosome formation and transport.

Acetylsalicylic acid (aspirin; ASA) is widely used as an analgesic/antipyretic drug. ASA exhibits a wide range of biological effects, including preventative effects against heart attack, stroke, and the development of some types of cancer. However, the effects of ASA on melanogenesis are not well known. Therefore, we investigated the effect of ASA on melanin production using B16 murine melanoma cells and demonstrated a new biological effect of ASA. In the presence of alpha-melanocyte stimulating hormone (alpha-MSH), B16 melanoma cells are stimulated to enhance melanin synthesis. ASA (2 mM) inhibited alpha-MSH-enhanced melanin synthesis in melanoma more strongly than other well-known anti-melanogenic agents such as arbutin (2 mM) and kojic acid (200 microM). Interestingly, ASA did not inhibit the catalytic activity of mushroom tyrosinase (concentration range 0.5-4.0 mM). To clarify the target of ASA action in melanogenesis, we performed Western blotting for tyrosinase, which is a key melanogenic enzyme. ASA inhibited tyrosinase expression in a dose-dependent manner. Therefore, the depigmenting effect of ASA might be due to inhibition of tyrosinase expression or enhancement of tyrosinase degradation. This study suggests that ASA is a candidate anti-melanogenic agent and it might be effective in hyperpigmentation disorders.

[F-18] fluorodopa ([F-18] DOPA) accumulates in the synaptic terminal of the dopaminergic neuron depending on the enzyme activity converting dopa into dopamine. The enzyme activity can be up/down-regulated by disease conditions, while the number of dopamine transporter is thought to be defined by the number of the synapse. There are four major pathways of dopaminergic projection systems. The nigrostriatal pathway is particularly involved in the production of movement, as part of a system called the basal ganglia motor loop. The mesocortila/ limbic pathway is be involved in cognitive function and motivation and emotional response. Dopaminergic functions in the extrastriatal area in addition to the striatum in vivo have been visualized with the combination of [F-18] DOPA PET and statistical image analyses. Ito K found the significant differences of influx rate (Ki) calculated with voxel-by-voxel Patlak analysis among Parkinson's disease (PD), PD with dementia (PDD), and normal control. Compared with the normal group, SPM localized declines of the [F-18] DOPA Ki bilaterally in the putamen, the right caudate nucleus and the left ventral midbrain for the PD group. Compared with the normal group, the PDD group showed reduced [F-18] DOPA Ki bilaterally in the striatum, midbrain and anterior cingulate. A relative difference in 18F-dopa uptake between PD and PDD was the bilateral decline in the anterior cingulate area and ventral striatum and in the right caudate nucleus in the PDD group. Accordingly, we conclude that dementia in PD is associated with impaired mesolimbic and caudate dopaminergic function. The next question is whether the corresponding dopaminergic change exists in the neural ganglia in the midbrain. We developed a method optimaized for the statistical analysis of the brain stem. PD showed slight increase of Ki in the raphe nucleus and the locus ceruleus. In contrast, PDD demonstrated decline tendency of Ki in the raphe and the locus ceruleus. These suggest cognitive impairment in PDD is caused by the affected the mesolimbic dopaminergic system which originates in the ventral tegmental area. This finding corresponds to Braak's staging of the intracerebral inclusion body pathology associated with PD.

In this work, a novel strategy was constructed to determine the enantiomeric composition of chiral substances discriminated by bovine serum albumin (BSA) based on the UV-vis spectra of the receptor-ligand mixtures coupled with partial least squares (PLS-1) analysis. Taking tryptophan (Trp) enantiomer as an example, when 20 microM BSA was used, the enantiomeric composition was accurately determined with concentration of only 100 nM and the corresponding enantiomeric excess as high as 98% (or -98%), which is relatively more sensitive than in literature. Furthermore, the BSA-based approach was also used to predict the enantiomeric composition of other chiral compounds, such as phenylalanine (Phe), tyrosine (Tyr), alanine (Ala), cysteine (Cys), DOPA and propranolol (Prop). The results fully demonstrate that BSA is effective in determination of enantiomeric composition of some chiral compounds.

Tyrosine levels do not generally affect indices of dopamine (DA) synthesis or efflux under basal conditions, but can do so when DA synthesis is increased. One possibility is that a high rate of DA synthesis depletes the normally adequate pool of endogenous tyrosine. To study this, we administered drugs known to preferentially increase striatal DA synthesis and examined DOPA levels in striatal microdialysate during perfusion with NSD-1015. In additional groups, we also measured DA, tyrosine and large neutral amino acids in striatal microdialysate, as well as in tissue from striatum and medial prefrontal cortex (MPFC). gamma-butyrolactone (GBL) (750 mg/kg i.p.) increased DOPA levels in striatal microdialysate, increased tissue DA levels in the MPFC and striatum, but lowered tissue tyrosine levels only in striatum. In striatal microdialysate, GBL markedly lowered DA levels; tyrosine levels were only marginally lower. Haloperidol (HAL) (1.0 mg/kg s.c.)+/-amfonelic acid (AFA) (5 mg/kg i.p.) increased striatal DOPA accumulation, increased striatal DA efflux, lowered striatal tissue tyrosine levels, but did not affect microdialysate tyrosine levels. There were no consistent changes in levels of other large neutral amino acids. We conclude that increased tyrosine hydroxylation can significantly deplete the endogenous pool of tyrosine. Under such conditions, near normal extracellular tyrosine levels are maintained despite lower tissue levels. The data are consistent with a net transfer of tyrosine from non-DAergic cells to DA terminals in support of DA synthesis.

Previous studies have provided compelling evidence for the presence of oxidized proteins and lipids in advanced human atherosclerotic lesions. The catalyst responsible for such oxidation is unknown and controversial. We have previously provided evidence for elevated levels of iron in lesions. In this study we hypothesized that if iron ions catalyzed protein and lipid oxidation in the artery wall, then there should be a positive correlation between these parameters. Iron concentrations in ex vivo healthy human arteries and advanced carotid lesions were quantified by electron paramagnetic resonance spectroscopy. Four specific side-chain oxidation products of proteins, and the lipid oxidation products 7-ketocholesterol and cholesterol ester alcohols and hydroperoxides, were quantified by HPLC in the same samples used for the iron measurements. Parent amino acids, cholesterol, and cholesterol esters were also quantified. Statistically elevated levels of iron, cholesterol, cholesterol esters, 7-ketocholesterol, and cholesterol ester alcohols and hydroperoxides were detected in advanced lesions compared with healthy control tissue. Iron levels correlated positively and strongly with all four markers of protein oxidation, but not with either marker of lipid oxidation. These data support the hypothesis that elevated levels of iron contribute to the extent of protein, but not lipid, oxidation in advanced human lesions.

Previous studies have examined the conjugation of sulfhydryl compounds such as L-cysteine and glutathione with DOPA-quinone following the oxidation of tyrosine and DOPA by tyrosinase. These covalent reactions play a key role in the regulation and metabolism of pigment cells. We report on the first direct evidence for the formation of lipoyl adducts in reactions of thiol groups with DOPA-quinone in dihydrolipoic acid (6,8-dimercaptooctanoic acid [DHLA]). Incubating DHLA with DOPA-quinone followed by tyrosinase-catalyzed oxidation resulted in the three products predicted by HPLC-UV and LC-ESI(-)-MS analyses for DHLA DOPA conjugates. In the current study, we identified 5-S-lipoyl-DOPA among the principal products isolated by HPLC and characterized by FAB(-)-MS, ESI(-)-MS/MS, and 1H NMR, 2D-COSY studies. Collectively, these results suggest that DHLA undergoes sulfhydryl conjugation with DOPA-quinone, pointing to the involvement of thiol-reactive metabolites.

The purpose of the present study was to determine whether the activation of NPY receptors alters catecholamines (CA) synthesis in the central nervous system and, if so, to identify the NPY receptor subtype(s) mediating this effect. Tyrosine hydroxylation, the rate-limiting step in CA synthesis, was assessed by measuring the accumulation of 3,4-dihydroxyphenyalanine (DOPA) by high pressure liquid chromatography coupled to electrochemical detection (HPLC-EC) in rat striatal dices following incubation of the tissue with the aromatic L-amino acid decarboxylase inhibitor m-hydroxybenzyl hydrazine (NSD 1015). Treatment with NSD 1015 resulted in an increase in DOPA accumulation that was increased even further following depolarization with a high potassium (KCl) buffer. PYY13-36 and NPY13-36 both produced a significant enhancement of the KCl-induced increase in DOPA accumulation. The effect of PYY13-36 was completely attenuated by the selective Y2 antagonist BIIE0246 suggesting that activation of Y2 receptors enhanced the synthesis of dopamine. In contrast to the effects of NPY13-36 and PYY13-36; NPY, PYY and PYY3-36 all produced a significant attenuation of the KCl-induced increase in DOPA accumulation. The Y1 antagonist BIBO3304 and the Y5-antagonist CGP71683A, both prevented the inhibitory effect of NPY converting it to a stimulatory effect. The enhancement of the NPY induced increase in DOPA accumulation observed by BIBO3304 was attenuated when examined in the presence of the Y2 antagonist BIIE0246. These results suggest that activation of NPY receptors can modulate the synthesis of CA in the rat striatum. The Y1 and Y5 receptor appear to be involved in attenuation, while Y2 receptors are involved in the stimulation of synthesis.

Silver-staining procedures were investigated for their effectiveness in identifying cell wall-based fungal melanins in live and fixed plastic embedded samples, particularly 1,8-dihydroxynaphthalene (DHN) based polyketide melanins. We developed a simple and reliable melanin-staining technique based on a silver accumulation method originally published for histological demonstration of heavy metal sulfides in mammalian tissues. Copper is bound to fungal melanin followed by formation of the copper sulfide at melanin sites in fungal cell walls, which then are amplified into vivid black stains using a silver enhancement step. The method demonstrates patterns of melanization in a range of fungal hyphae and is suitable for light and electron microscopy. Albino mutant fungi and normally nonmelanized fungi do not stain with the sulfide-silver technique. Mammalian melanocytes also were labeled by the technique, indicating its universality as a melanin probe.

The amino acid sequence of methylamine oxidase (MeAO) from the fungus Aspergillus niger was analyzed using mass spectrometry (MS). First, MeAO was characterized by an accurate molar mass of 72.4 kDa of the monomer measured using MALDI-TOF-MS and by a pI value of 5.8 determined by isoelectric focusing. MALDI-TOF-MS revealed a clear peptide mass fingerprint after tryptic digestion, which did not provide any relevant hit when searched against a nonredundant protein database and was different from that of A. niger amine oxidase AO-I. Tandem mass spectrometry with electrospray ionization coupled to liquid chromatography allowed unambiguous reading of six peptide sequences (11-19 amino acids) and seven sequence tags (4-15 amino acids), which were used for MS BLAST homology searching. MeAO was found to be largely homologous to a hypothetical protein AN7641.2 (EMBL/GenBank protein-accession code EAA61827) from Aspergillus nidulans FGSC A4 with a theoretical molar mass of 76.46 kDa and pI 6.14, which belongs to the superfamily of copper amine oxidases. The protein AN7641.2 is only little homologous to the amine oxidase AO-I (32% identity, 49 % similarity).

Protein-bound 3,4-dihydroxyphenylalanine (PB-DOPA) is a major product of hydroxyl radical attack on tyrosine residues of proteins. Levels of PB-DOPA in cells and tissues have been shown to be greatly elevated in age-related diseases. We demonstrate for the first time that l-DOPA (levodopa) can be biosynthetically incorporated into cell proteins by human cells (THP-1 monocytes and monocyte-derived macrophages). The DOPA-containing proteins generated were selectively visualized on PVDF membranes using a redox-cycling staining method. Many cell proteins contained DOPA and seemed to be synthesized as their full-length forms. The cellular removal of DOPA-containing proteins by THP-1 cells was by proteolysis involving both the proteasomal and the lysosomal systems. The rate of cellular proteolysis of DOPA-containing proteins increased at lower levels of DOPA incorporation but decreased at higher levels of DOPA incorporation. The decreased rate of degradation was accompanied by an increase in the activity of cathepsins B and L but the activity of cathepsin S increased only at lower levels of DOPA incorporation. These data raise the possibility that PB-DOPA could be generated in vivo from l-DOPA, which is the most widely used treatment for Parkinson disease.

The electrospray ionization (ESI) voltage is shown to interfere with liquid chromatographic separations performed with packed porous graphitic carbon (PGC) capillary columns. This interference is ascribed to the presence of an electric field over the conductive column in the absence of an earth point between the column and the ESI emitter. The current evolved alters the chromatographic behavior of the catecholamine metabolite 3-O-methyl-DOPA significantly, as both peak splitting and a dramatic decrease in the retention time were observed. Furthermore, the response from the mass spectrometer was decreased by 33% at the same time. A related compound, tyrosine, exhibited decreased retention times but no peak splitting, whereas no shifts in the retention times (or peak splitting) were seen for the less retained dopamine and noradrenaline. When the current through the PGC column was eliminated by the use of an earth point between the column and the ESI emitter, the chromatographic behavior of the column was found to return slowly to normal after hours of equilibration with 60 : 40 (v/v) methanol-ammonium formate buffer of pH 2.9. The behavior of the PGC column with and without the earth point was found to be highly reproducible during a period of 1 month. We propose that the effect of the ESI voltage on the chromatographic behavior of the PGC column is due to associated redox reactions affecting both the PGC particles and the analytes. It is concluded that (for analytical reasons), care should be taken to ensure that no current is flowing through the chromatographic system when interfacing PGC columns, and conducting parts in general, to ESI mass spectrometry., (Copyright 2004 John Wiley & Sons, Ltd.)

Introduction: Protein-bound dihydroxyphenylalanine (PB-DOPA) and its oxidation products are formed by free radical and oxidative attack on proteins. Hemodialysis and uremic toxins can activate leukocytes leading to overproduction of reactive oxygen species such as hydrogen peroxide and hypochlorous acid (HOCl) that increases protein oxidation., Methods: We have used a sensitive fluorometric method to measure PB-DOPA and its oxidation products in proteins after gamma-irradiation and incubation with HOCl and in serum from hemodialysis patients and healthy controls. These PB-DOPA concentrations were compared with those measured by HPLC (PB-DOPAHPLC)., Results: Fluorescent PB-DOPA increased linearly with increasing amounts of human serum and with increasing amounts of gamma-irradiated bovine serum albumin. Concentrations of fluorescent PB-DOPA paralleled PB-DOPAHPLC levels but were approximately 60-70 times higher. Incubation of BSA and human serum albumin (HSA) with HOCl (39.4 mM) significantly (P < 0.0001) increased fluorescent PB-DOPA by 5 fold and 10 fold respectively and PB-DOPAHPLC by 6-fold for both proteins Fluorescent PB-DOPA concentration increased significantly (P < 0.0001) by 16-fold in human serum incubated with HOCl (39.4 mM). Mean serum fluorescent PB-DOPA was significantly (P < 0.0001) higher in 19 hemodialysis patients (57.7 +/- 16.1 microM) compared with 21 healthy controls (33.5 +/- 3.7 microM). Mean PB-DOPAHPLC was 4.45 +/- 1.63 microM in the healthy controls and 12 hemodialysis patients had values within the range of values in these controls while five patients had values that were outside eight SDs of the mean for healthy subjects. Serum fluorescent PB-DOPA was not correlated significantly with PB-DOPAHPLC in these subjects., Conclusions: The results of this study suggest that fluorophores of the type, which are derived from DOPA can be reproducibly measured in delipidated serum protein and that HOCl can increase levels of these fluorophores-generating proteins and may potentially contribute to the high levels in serum from hemodialysis patients. This high level of fluorescent PB-DOPA compounds is only partially due to authentic PB-DOPA and might also be derived from other related protein oxidation products including those from DOPA oxidation.

The oocyst wall of apicomplexan parasites protects them from the harsh external environment, preserving their survival prior to transmission to the next host. If oocyst wall formation could be disrupted, then logically, the cycle of disease transmission could be stopped, and strategies to control infection by several organisms of medical and veterinary importance such as Eimeria, Plasmodium, Toxoplasma, Cyclospora, and Neospora could be developed. Here, we show that two tyrosine-rich precursor glycoproteins, gam56 and gam82, found in specialized organelles (wall-forming bodies) in the sexual stage (macrogamete) of Eimeria maxima are proteolytically processed into smaller glycoproteins, which are then incorporated into the developing oocyst wall. The identification of high concentrations of dityrosine and 3,4-dihydroxyphenylalanine (DOPA) in oocyst extracts by high-pressure liquid chromatography, together with the detection of a UV autofluorescence in intact oocysts, implicates dityrosine- and possibly DOPA-protein cross-links in oocyst wall hardening. In addition, the identification of peroxidase activity in the wall-forming bodies of macrogametes supports the hypothesis that dityrosine- and DOPA-mediated cross-linking might be an enzyme-catalyzed event. As such, the mechanism of oocyst wall formation in Eimeria, is analogous to the underlying mechanisms involved in the stabilization of extracellular matrices in a number of organisms, widely distributed in nature, including insect resilin, nematode cuticles, yeast cell walls, mussel byssal threads, and sea urchin fertilization membranes.