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The aim of the research was to compare the impact of nano- and micro-sized-zinc on the kinetics of changes in the level of 3-methyladenine, 7-methylguanine, 7-methylguanosine, O-methylguanosine, 1-methyladenosine, N6-methyl-2’-deoxyguanosine in urine of rats with breast cancer. Female Sprague-Dawley rats divided into 3 groups were used in the study. Animals were fed only a control diet or diets supplemented with the nano and micro-sized zinc particles. To induce the mammary cancer (adenocarcinoma), rats were treated with 7,12-dimethylbenz[a]anthracene (DMBA). Modified nucleosides were determined by a validated high performance liquid chromatography coupled to mass spectrometry method. In the first stage of investigations a synergistic activity of nanosized Zn with DMBA on the growth of the neoplastic process was found. During that time a statistically significant increase in the levels of all six examined markers in the rats’ urine was observed. However, as the experiment continued, the supplementation with nanosized zinc caused inhibition of tumour growth, being followed by regression and remission of tumours, as well as, a statistically significant systematic reduction of the levels of methyl derivatives in the urine. Biopsy images indicated grade 1 tumours with multiple inflammatory infiltrates in the group treated with zinc nanoparticles, whereas, in the other groups, moderately-differentiated grade 2 adenocarcinoma was identified. It was found that the biological activity of zinc depends on the size of applied particles, as the treatment with zinc microparticles has not had much effect on cancer progression.

The endocrine activity of the thyroid gland is accomplished by its follicular and parafollicular cells. In these cells, numerous G proteinsdependent pathways are active and potentially could be regulated by a 33-kDa cytoplasmic protein phosducin, which interacts with the Gβ subunit and may compete with Gα or Gβγ dimer effectors. Significant expression of phosducin has been shown in the retina, pineal gland, and some neurons. Here, we studied postoperative thyroid tissue samples collected from patients with nodular goiter and 2 thyroid-derived cell lines for the presence of phosducin. Using reverse transcription PCR with product sequencing and highly sensitive immunodetection we identified phosducin mRNA and protein in the thyroid gland and parafollicular C TT cells, but not in the follicular Nthy-ori 3-1 cell line. We also observed that siRNA-mediated silencing of phosducin gene expression decreased Ca2+-stimulated secretion of calcitonin and serotonin by TT cells. [ABSTRACT FROM AUTHOR]

Despite advancements in early detection and treatment in developed countries, colorectal cancer (CRC) remains the third most common malignancy and the second-leading cause of cancer-related deaths worldwide. Conventional chemotherapy, a key option for CRC treatment, has several drawbacks, including poor selectivity and the development of multiple drug resistance, which often lead to severe side effects. In recent years, the use of polysaccharides as drug delivery systems (DDSs) to enhance drug efficacy has gained significant attention. Among these polysaccharides, chitosan (CS), a linear, mucoadhesive polymer, has shown promise in cancer treatment. This review summarizes current research on the potential applications of CS-based hydrogels as DDSs for CRC treatment, with a particular focus on smart hydrogels. These smart CS-based hydrogel systems are categorized into two main types: stimuli-responsive injectable hydrogels that undergo sol-gel transitions in situ, and single-, dual-, and multi-stimuli-responsive CS-based hydrogels capable of releasing drugs in response to various triggers. The review also discusses the structural characteristics of CS, the methods for preparing CS-based hydrogels, and recent scientific advances in smart CS-based hydrogels for CRC treatment.

Rapid development of tissue engineering in recent years has increased the importance of three-dimensional (3D) bioprinting technology as novel strategy for fabrication functional 3D tissue and organoid models for pharmaceutical research. 3D bioprinting technology gives hope for eliminating many problems associated with traditional cell culture methods during drug screening. However, there is a still long way to wider clinical application of this technology due to the numerous difficulties associated with development of bioinks, advanced printers and in-depth understanding of human tissue architecture. In this review, the work associated with relatively well-known extrusion-based bioprinting (EBB), jetting-based bioprinting (JBB), and vat photopolymerization bioprinting (VPB) is presented and discussed with the latest advances and limitations in this field. Next we discuss state-of-the-art research of 3D bioprinted in vitro models including liver, kidney, lung, heart, intestines, eye, skin as well as neural and bone tissue that have potential applications in the development of new drugs., 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 © 2023 Elsevier B.V. All rights reserved.)

Camptothecin (CPT), an alkaloid with potent anticancer activity, is still not used in clinical practice due to its high hydrophobicity, toxicity, and poor active-form stability. To address these shortcomings, our research focuses on the encapsulation of this drug in the poly(amidoamine) (PAMAM) dendrimer macromolecule. The PAMAM dendrimer/CPT complex was synthesized and thoroughly characterized. The in vitro drug release study revealed that the drug was released in a slow and controlled manner in acidic and physiological conditions and that more than 80% of the drug was released after 168 h of incubation. Furthermore, it was demonstrated that CPT was released with first-order kinetics and non-Fickian transport. The studies on the hemolytic activity of the synthesized complex indicated that it is hemocompatible for potential intravenous administration at a concentration ≤ 5 µg/mL. Additionally, the developed product was shown to reduce the viability of non-small-cell lung cancer cells (A549) in a concentration- and time-dependent manner, and cancer cells were more susceptible to the complex than normal fibroblasts. Lastly, molecular modeling studies revealed that the lactone or carboxylic forms of CPT had a significant impact on the shape and stability of the complex and that its formation with the lactone form of CPT was more energetically favorable for each subsequent molecule than the carboxylic form. The report represents a systematic and structured approach to develop a PAMAM dendrimer/CPT complex that can be used as an effective drug delivery system (DDS) for the potential treatment of non-small-cell lung cancer.

Camptothecin (CPT) has demonstrated antitumor activity in lung, ovarian, breast, pancreas, and stomach cancers. However, this drug, like many other potent anticancer agents, is extremely water-insoluble. Furthermore, pharmacology studies have revealed that prolonged schedules must be administered continuously. For these reasons, several of its water-soluble analogues, prodrugs, and macromolecular conjugates have been synthesized, and various formulation approaches have been investigated. Biodegradable polyesters have gained popularity in cancer treatment in recent years. A number of biodegradable polymeric drug delivery systems (DDSs), designed for localized and systemic administration of therapeutic agents, as well as tumor-targeting macromolecules, have entered clinical trials, demonstrating the importance of biodegradable polyesters in cancer therapy. Biodegradable polyester-based DDSs have the potential to deliver the payload to the target while also increasing drug availability at intended site. The systemic toxicity and serious side-effects associated with conventional cancer therapies can be significantly reduced with targeted polymeric systems. This review elaborates on the use of biodegradable polyesters in the delivery of CPT and its analogues. The design of various DDSs based on biodegradable polyesters has been described, with the drug either adsorbed on the polymer's surface or encapsulated within its macrostructure, as well as those in which a hydrolyzed chemical bond is formed between the active substance and the polymer chain. The data related to the type of DDSs, the kind of linkage, and the details of in vitro and in vivo studies are included.

Hybrid nanostructures can be developed with inorganic nanoparticles (NPs) such as zinc oxide (ZnO) and natural antibacterials. ZnO NPs can also exert antibacterial effects, and we used them here to examine their dual action in combination with a natural antibacterial agent, protocatechuic acid (PCA). To produce hybrid nanoformulations, we functionalized ZnO NPs with four types of silane organic molecules and successfully linked them to PCA. Physicochemical assessment confirmed PCA content up to ~18% in hybrid nanoformulations, with a PCA entrapment efficiency of ~72%, indicating successful connection. We then investigated the in vitro release kinetics and antibacterial effects of the hybrid against Staphylococcus aureus . PCA release from hybrid nanoformulations varied with silane surface modification. Within 98 h, only 8% of the total encapsulated PCA was released, suggesting sustained long-term release. We used nanoformulation solutions collected at days 3, 5, and 7 by disc diffusion or log reduction to evaluate their antibacterial effect against S. aureus . The hybrid nanoformulation showed efficient antibacterial and bactericidal effects that also depended on the surface modification and at a lower minimum inhibition concentration compared with the separate components. A hybrid nanoformulation of the PCA prodrug and ZnO NPs offers effective sustained-release inhibition of S. aureus growth.

Hydroxyapatite (HA) powders enriched with silver or gallium ions or both were synthesized by two different routes: standard precipitation and the solid-state method. The powders were characterized by using several methods: inductively coupled plasma optical emission spectrometry (ICP-OES), powder X-ray diffractometry (PXRD), transmission electron microscopy (TEM), infrared spectroscopy (FT-IR) and solid-state nuclear magnetic resonance spectroscopy (ssNMR). The effects of enrichment of the HAs in Ag + or Ga 3+ or both on in vitro cytotoxicity and microbiological activity were discussed. PXRD experiments showed that the samples obtained by the wet method consisted of single-phase nanocrystalline HA, while the samples prepared via the solid-state method are microcrystalline with a small amount of calcium oxide. The introduction of higher amounts of silver ions was found to be more effective than enriching HA with small amounts of Ag + . Gallium and silver ions were found not to affect the lattice parameters. Ga 3+ affected the crystallinity of the samples as well as the content of structural hydroxyl groups. Among samples synthesized by the wet method, only one (5Ag-HAw) was cytotoxic, whereas all Ga-containing samples obtained by the dry method showed cytotoxicity. In the preliminary antimicrobial test all the materials containing "foreign" ions showed high antibacterial activity.

Cationic antimicrobial peptides represent a promising therapeutic option against multidrug-resistant bacteria for the treatment of local infections. However, due to their low stability and potential toxicity, there are limited possibilities for their application in clinical practice. In this study, different poly(ε-caprolactone) (PCL) microparticles (MPs) loaded with citropin 1.1 (CIT) were investigated in order to demonstrate the effect of the polymer microstructure on the encapsulation efficiency ( EE ) and kinetics of the peptide release from the newly developed devices. The characteristics of the new systems in terms of surface morphology, particle size, EE and zeta potential analysis, as well as the haemolytic activities of the peptide were investigated. The in vitro release kinetics of CIT from the MPs was also investigated. CIT loading was favoured by a high content of negative charged linear polymer chains in the PCL structure. The presence of non-charged, amorphous macrocycle domains results in faster degradation of the PCL matrix. Depending on the crystallinity of the PCL, the peptide release exhibited a near-zero-order or near-first-order profile with no “burst release”. The results indicated that CIT-loaded PCL MPs could potentially be a promising drug delivery system (DDS) for the treatment of local infections.

Antimicrobial peptides (AMPs) are prospective therapeutic options for treating multiple-strain infections. However, clinical and commercial development of AMPs has some limitations due to their limited stability, low bioavailability, and potential hemotoxicity. The purpose of this study was to develop new polymeric carriers as highly controlled release devices for amphibian peptides citropin 1.1 (CIT) and temporin A (TEMP). The release rate of the active pharmaceutical ingredients (APIs) was strongly dependent on the API characteristics and the matrix microstructure. In the current work, we investigated the effect of the polymer microstructure on in vitro release kinetics of AMPs. Non-contact laser profilometry, scanning electron microscopy (SEM), and differential scanning calorimetry (DSC) were used to determine the structural changes during matrix degradation. Moreover, geno- and cytotoxicity of the synthesized new carriers were evaluated. The in vitro release study of AMPs from the obtained non-toxic matrices shows that peptides were released with near-zero-order kinetics. The peptide "burst release" effect was not observed. New devices have reached the therapeutic concentration of AMPs within 24 h and maintained it for 28 days. Hence, our results suggest that these polymeric devices could be potentially used as therapeutic options for the treatment of local infections.

Micro-organism resistance is an important challenge in modern medicine due to the global uncontrolled use of antibiotics. Natural and synthetic antimicrobial peptides (AMPs) symbolize a new family of antibiotics, which have stimulated research and clinical interest as new therapeutic options for infections. They represent one of the most promising antimicrobial substances, due to their broad spectrum of biological activity, against bacteria, fungi, protozoa, viruses, yeast and even tumour cells. Besides, being antimicrobial, AMPs have been shown to bind and neutralize bacterial endotoxins, as well as possess immunomodulatory, anti-inflammatory, wound-healing, angiogenic and antitumour properties. In contrast to conventional antibiotics, which have very defined and specific molecular targets, host cationic peptides show varying, complex and very rapid mechanisms of actions that make it difficult to form an effective antimicrobial defence. Importantly, AMPs display their antimicrobial activity at micromolar concentrations or less. To do this, many peptide-based drugs are commercially available for the treatment of numerous diseases, such as hepatitis C, myeloma, skin infections and diabetes. Herein, we present an overview of the general mechanism of AMPs action, along with recent developments regarding carriers of AMPs and their potential applications in medical fields., (© 2017 John Wiley & Sons A/S.)

To evaluate the effects of ionic liquids (ILs) on the microstructural features of aliphatic polyesters for biomedical applications, a series of copolymers were synthesized by lipase ring opening polymerization of rac -lactide ( rac -LA) and ε-caprolactone (CL). The chemical structures of resulting polymers were characterized by ¹H- and 13 C-NMR and the average molecular weight ( M n ) and dispersity index were characterized by gel permeation chromatography. The structure of the copolymers confirms the presence of linear polymer chains with end-functional hydroxyl groups allowing covalent coupling of the therapeutic agents. Chain microstructure of copolymers indicates the presence of both random and block copolymers depending on the synthesis conditions. Moreover, it was found that CL is the most active co-monomer during copolymerization which enhances the polymerizability of rac -LA and allows to obtain higher M n of the copolymers. The results demonstrate that ILs could be promising solvents in synthesis of aliphatic esters for biomedical applications.

Ag + -substituted hydroxyapatites (Ag-HAs) and Ag + /CO 3 2- -co-substituted hydroxyapatites (Ag-CHAs) with two different concentrations of silver ions were synthesized by the standard precipitation method. For comparison, pure hydroxyapatite (HA) and carbonated hydroxyapatite (CHA) were synthesized using the same method. The obtained powders were examined by various physicochemical methods, such PXRD, TEM, FTIR and ssNMR. Elemental analysis was provided by WD-XRF and ICP-MS methods. The strains of Staphylococcus aureus and Escherichia coli were used to evaluate the antibacterial activity of the materials. The study demonstrates that the substituted samples are homogenous and poorly crystalline. Introducing carbonates into the crystal structure significantly affects the physicochemical properties of the silver containing hydroxyapatite, i.e., crystallinity, lattice parameters, crystal size and morphology or content of structural hydroxyl groups. Carbonate substitution leads to a significant increase in the contribution of the hydrated surface layer. Ag-CHA nanocrystals with an enhanced hydrated surface layer and higher solubility demonstrate an admirable antibacterial effect., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Purpose: The purpose of this study was to develop the perspective biodegradable poly(ester-urethane) (PUR) carriers based on "predominantly isotactic" and atactic polylactides (PLAs), and poly(ε-caprolactone) (PCL), for the controlled release of epirubicin (EPI)., Methods: The biodegradable PURs containing different soft segments as new and effective carriers of EPI have been obtained. The preliminary studies on toxicity and degradation of obtained polymers, and the release of the EPI from PUR carriers were carried out., Results: We found that the kinetic release of EPI from the obtained PUR carriers tested in vitro at 37°C and pH 7.4 was strongly dependent on the kind of the polyesters, used as the soft segment in PURs synthesis. Furthermore, we demonstrated that the EPI was released from various synthesized carriers in a rather regular manner, according to the diffusion-degradation and degradation mechanisms. Importantly, in some cases, the kinetics of the EPI release was nearly zero-order., Conclusion: The results show that the obtained PURs are very effective and perspective carriers and might be potentially applied in the technology of high controlled EPI delivery systems.

Polylactide (PLA) represents one of the most promising biomedical polymers due to its biodegradability, bioresorbability and good biocompatibility. This work highlights the synthesis and characterization of PLAs using novel diethylzinc/gallic acid (ZnEt₂/GAc) and diethylzinc/propyl gallate (ZnEt₂/PGAc) catalytic systems that are safe for human body. The results of the ring-opening polymerization (ROP) of rac-lactide (rac-LA) in the presence of zinc-based catalytic systems have shown that, depending on the reaction conditions, "predominantly isotactic", disyndiotactic or atactic PLA can be obtained. Therefore, the controlled and stereoselective ROP of rac-LA is discussed in detail in this paper.

In this work, we used the co-precipitation method to synthesize hydroxyapatite (Mn-SeO3-HA) containing both selenium IV (approximately 3.60 wt.%) and manganese II (approximately 0.29 wt.%). Pure hydroxyapatite (HA), hydroxyapatite-containing manganese (II) ions (Mn-HA), and hydroxyapatite-containing selenite ions alone (SeO3-HA), prepared with the same method, were used as reference materials. The structures and physicochemical properties of all the obtained samples were investigated. PXRD studies showed that the obtained materials were homogeneous and consisted of apatite phase. Introducing selenites into the hydroxyapatite crystals considerably affects the size and degree of ordering. Experiments with transmission electron microscopy (TEM) showed that Mn-SeO3-HA crystals are very small, needle-like, and tend to form agglomerates. Fourier transform infrared spectroscopy (FT-IR) and solid-state nuclear magnetic resonance (ssNMR) were used to analyze the structure of the obtained material. Preliminary microbiological tests showed that the material demonstrated antibacterial activity against Staphylococcus aureus, yet such properties were not confirmed regarding Escherichia coli. PACS codes: 61, 76, 81.

Biodegradable or bioresorbable polymers are commonly used in various pharmaceutical fields (e.g., as drug delivery systems, therapeutic systems or macromolecular drug conjugates). Polyesters are an important class of polymers widely utilized in pharmacy due to their biodegradability and biocompatibility features. In recent years, there has been increased interest in enzyme-catalyzed ring-opening polymerization (e-ROP) of cyclic esters as an alternative method of preparation of biodegradable or bioresorbable polymers. Ionic liquids (ILs) have been presented as green solvents in enzymatic ring-opening polymerization. The activity, stability, selectivity of enzymes in ILs and the ability to catalyze polyester synthesis under these conditions are discussed. Overall, the review demonstrates that e-ROP of lactones or lactides could be an effective method for the synthesis of useful biomedical polymers.

The functioning of heterotrimeric G protein α subunits in the transduction of hormonal signals to appropriate intracellular responses is well recognized. Much less is known about the distribution of isoforms and functions of G protein β subunits. Here, using specific antibodies, we documented that in plasma membranes of the thyroid cell line Nthy-ori 3-1 all Gβ isoforms-Gβ(1), Gβ(2), Gβ(3), Gβ(4) and Gβ(5) are present, while the Gβ(3) occurs in minute amount. In plasma membrane fraction isolated from pooled postoperative thyroids of patients with nodular goiter and Graves' disease, the Gβ(1), Gβ(2), Gβ(4) and Gβ(5) subunits were found, whereas Gβ(3) could not be detected. Competition studies revealed that the Gβ(2) is the principal Gβ subunit in membranes from cultured thyroid cells, originated from normal thyroid, as well as in membranes from patients' thyroids. This suggests that Gβ(2) subunit cooperates with Gα(s) subunit, the most active of the Gα variants, during stimulation of adenylate cyclase which constitutes the main route of physiological thyroid stimulation., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Phosducin family proteins are regulators of cytoplasmic processes. The main function ascribed to phosducin is the binding and sequestration of the β subunit of heterotrimeric G proteins. Phosducin-like protein 1, longer than phosducin by 37 amino-acids, is involved in chaperoning of newly synthesized proteins. β-Actin, a component of the cytoskeleton, participates in cell movement. There is no apparent evolutionary relationship between phosducin and β-actin nor structure similarity. Nevertheless we obtained the polyclonal antibodies named ap33, originally directed against a phosducin-derived peptide (SQSLEEDFEGQATHTGPK), that also recognized β-actin. The epitope on the β-actin molecule was characterized. It is a conformational epitope grouping some of the L-D-F-E-Q-A-T-K amino-acids found in the peptide originally used to obtain the antibodies. The main part of the epitope is localized on the actin-actin interface of polymerized actin, so it is accessible only on monomeric actin. The existence of a common epitope on the molecules of phosducin and β-actin may reflect a topological similarity of a small region of their surfaces., (Copyright © 2010 Elsevier B.V. All rights reserved.)

In a previous study, we noticed an unexpected reaction of an antithyrotropin receptor ectodomain (ETSHR)-reactive monoclonal antibody, A10, with alpha1-antitrypsin (antitrypsin). Presently, we decided to probe the structural basis of this cross-reactivity. Recombinant ETSHR, antitrypsin, synthetic peptides corresponding to the region of similarity in these proteins (EEDFRV and EEDFHV, respectively) and a set of peptides related to this region, N- and C-terminally elongated, were used in the study. Comparing the values of the dissociation constants, we found that the affinity of peptides corresponding to the region of similarity to monoclonal antibody A10 was the same in spite of a difference in one residue (R 38 in ETSHR and H 209 in antitrypsin), whereas a change of E 206 to R in antitrypsin-related peptide dramatically decreased the affinity. The whole binding site of A10 in ETSHR as well as in antitrypsin was larger than the region of similarity. We propose that residues ECHQEEDFV represent the monoclonal antibody A10 epitope. They form an almost continuous sequence of residues 30-37 and 39 in ETSHR. The monoclonal antibody A10 binding site on antitrypsin is shorter. It comprises amino acids 205-208 and 210, from the region of similarity with, probably, additional two residues, H-287 and E 363.

The majority of epitopes for TSH receptor (TSHR) stimulating autoantibodies are clustered around the Nterminal region of the TSH receptor. The characteristic feature of this region is the presence of four cysteine residues. It was proposed that cysteines in positions 29 and 41 in the receptor are connected by disulfide bonds and they are the target for receptor stimulating antibodies. The present study was aimed to check this possibility. The synthetic peptides: peptide corresponding to the part of TSHR containing the above 29-41 cysteine bond, the peptide similar to this peptide but without disulfide bond and the control peptide, containing sequence absent in the receptor were used for rabbit immunization. The thyroid status of all immunized rabbits was the same. Rabbits immunized with peptides related to TSHR generated antisera reactive with TSHR in immunoenzymatic assay. To check specificity of this reaction the influence of the peptides and the antisera on TSH binding to the receptor in competitive assay (TRAK) and their influence on adenylate cyclase activity were studied. It was found that neither synthetic peptides nor antiserum from any rabbit influenced TSH binding to the receptor in TRAK. In contrast low, but significant adenylate cyclase stimulating activity was noticed for antisera from two of six rabbit immunized by peptide containing the disulfide bond. We concluded that such a bond between cysteine residues 29 and 41 are present in TSHR in the site of stimulating antibodies epitope.

The thyrotropin receptor (TSHR) and alphal-antytripsin contain a fragment of sequence composed of 6 amino acids in which 5 residues are identical. Previously, we have suggested that this region of similarity [residues 34-39: (EEDFRV) in TSHR] is not the target for Graves' disease patients' autoantibodies. To verify this suggestion, we studied the reaction of patients' sera with alphal-antitrypsin. Two methods were used: TRAK assay, normally designed to estimate anti-TSHR autoantibodies in patients' sera, and immunoblotting. A modified version of the former assay was also used to study the influence of the synthetic peptide, corresponding to the region of similarity in TSHR, on Graves' patients sera or on thyrotropin (TSH) binding, and to study the influence of this peptide antiserum on TSH binding to the receptor. The TSHR stimulating and blocking activity of antisera to this peptide was studied in transfected Chinese hamster ovary cells. No influence of alphal-antitrypsin on the binding of patients' antibodies to the receptor were noticed nor were there reactions of autoantibodies with alphal-antitrypsin. We found that patients with anti-TSHR autoantibodies had a normal concentration of alphal-antitrypsin. A peptide corresponding to residues 34-39 did not influence Graves' patients sera and TSH binding and antiserum to this peptide did not influence TSH binding and adenylate cyclase activity. In summary, the results indicated that the sequence EEDFRV is not the target for patients autoantibodies.

The role of glycan moieties in thyrotropin receptor molecule in binding of antibodies is a subject of intense debate. To approach the function of sugars in recognition by antibody of the extracellular part of the receptor (ETSHR) we studied the reaction of the HPLC purified ETSHR from insect cells in the reaction with autoantibodies and antibodies of animal origin. None of the autoantibodies from Graves' patients sera bound to ETSHR. In contrast, each of the animal antibodies: three monoclonal, five polyclonal antireceptor and two polyclonal anti peptide corresponding to the amino acid sequence present in the receptor, became bound to the native receptor from insect cells as well as to its deglycosylated form. The shape of the dilution curves of particular antibodies in the reaction with either form of the receptor was almost the same. The coefficients of correlation was about 0.9. It seems that the correct receptor glycosylation is not crucial for binding of animal origin antibodies.

In a study designed to detect the presence of soluble, secreted A subunit of thyrotropin hormone receptor (TSHR) in serum, using anti-TSHR murine antibodies (mAbs) and peptide specific antiserum for Western blotting of human serum proteins fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) it was consistently observed that only one mAb, termed A10, reacted strongly with the 53 kd serum protein. The reaction was specific with the mAb A10 only, but not with another mAb or polyclonal antiserum. Furthermore, A10 immunoreactivity was documented in a variety of sera from healthy donors and patients, including patients whose thyroid gland was ablated during treatment for thyroid cancer. This suggests that the A10 cross-reactive protein was not derived from thyroid cells. The A10 cross-reactive protein was purified from normal serum and subjected to N-terminal sequence analysis, which identified the protein as alpha(1)-antitrypsin. Further experiments by enzyme-linked immunosorbent assay (ELISA) and the binding of antibody with deglycosylated or elastase-treated purified serum protein confirmed the cross-reactivity of mAb A10 with alpha(1)-antitrypsin. Alignment of the TSHR amino acid sequence with that of alpha(1)-antitrypsin identified five identical amino acids in a short stretch of residues 34-39 (EEDFRV) in TSHR and residues 205-210 (EEDFHV) in alpha(1)-antitrypsin. Analysis of the structural model of alpha(1)-antitrypsin revealed that these residues were exposed on the surface of alpha(1)-antitrypsin and were accessible for antibodies. Autoantibodies in patients with Graves' disease do not appear to recognize this region of the receptor and hence do not react with serum alpha(1)-antitrypsin.

The biosynthesis of thyroid hormone from thyroglobulin is catalysed by thyroid peroxidase (TPO), an integral membrane protein. TPO is also a major autoantigen in autoimmune thyroid disease and autoantibodies to TPO are markers for disease activity. Large quantities of purified TPO are essential for elucidating its structure and understanding its role in disease activity. We describe the high yield purification of full-length recombinant human TPO from baculovirus infected insect cells and compare it to purified native TPO from human thyroid glands. In contrast to native human TPO, the human TPO produced in insect cells as a recombinant protein was insoluble and resistant to solubilisation in detergents. Reversible substitution of lysine residues with citraconic anhydride led to increased solubility of the recombinant TPO, allowing high-yield purification by monoclonal antibody chromatography. The purified enzyme preparation was shown to be TPO by its reactivity with monoclonal and polyclonal antibodies by enzyme linked immunosorbent assay and Western blotting. Both the human and recombinant purified TPO preparations also react with sera from patients with autoimmune thyroid disease, although the binding of conformational dependent autoantibodies was considerably lower to the recombinant TPO than to the native TPO. This suggests that the recombinant TPO may differ in some aspects of its tertiary structure. The purified recombinant TPO was devoid of enzyme activity, in contrast to the enzymatically active, purified human TPO preparations. Both preparations contained comparable amounts of haem (R(z)=0.269), but a shift in the Soret band of recombinant TPO (402 nm) from that of natural TPO (409 nm) indicates that the lack of enzymatic activity of the recombinant enzyme may be due to changes in the protein backbone surrounding the haem. Both the purified native and recombinant TPO, under non-denaturing conditions, show evidence of high molecular mass oligomers, although the latter preparation is prone to a greater degree of aggregation. In conclusion, our studies indicate that recombinant TPO generated in insect cells is conformationally distinct from the native TPO, is insoluble and enzymatically inactive, consistent with the difficulties associated with its purification and crystallisation.