Optimized transformation, overexpression and purification of S100A10.

As a member of the S100 protein family, S100A10 has already been purified. However, its purity, or even yield, have often not been reported in the literature. To facilitate future biophysical experiments with S100A10, we aimed to obtain it at a purity of at least 95% in a reasonably large amount. Here, we report optimized conditions for the transformation, overexpression and purification of the protein. We obtained a purity of 97% and performed stability studies by circular dichroism. Our data confirmed that the S100A10 obtained is suitable for experiments to be performed at room temperature up to several days.

Isolation and Characterization of S100 Protein-Protein Complexes.

S100 proteins are small, mostly dimeric, EF-hand Ca2+-binding proteins. Upon Ca2+ binding, a conformational change occurs resulting in the exposure of a shallow hydrophobic binding groove in each subunit. Interestingly, S100 proteins can interact with their partners in two ways: symmetrically, when the two partners identically bind into each groove, or asymmetrically, when only one partner binds to the S100 dimer occupying both binding pockets. Here we present a heterologous expression and purification protocol for all known human S100 proteins as well as for their partner peptides. Moreover, we provide a detailed description of three in vitro methods to determine the affinity, stoichiometry, and kinetics of S100 protein-protein interactions.

Solving the crystal structure of human calcium-free S100Z: the siege and conquer of one of the last S100 family strongholds.

The X-ray structure of human apo-S100Z has been solved and compared with that of the zebrafish calcium-bound S100Z, which is the closest in sequence. Human apo-S100A12, which shows only 43% sequence identity to human S100Z, has been used as template model to solve the crystallographic phase problem. Although a significant buried surface area between the two physiological dimers is present in the asymmetric unit of human apo-S100Z, the protein does not form the superhelical arrangement in the crystal as observed for the zebrafish calcium-bound S100Z and human calcium-bound S100A4. These findings further demonstrate that calcium plays a fundamental role in triggering quaternary structure formation in several S100s. Solving the X-ray structure of human apo-S100Z by standard molecular replacement procedures turned out to be a challenge and required trying different models and different software tools among which only one was successful. The model that allowed structure solution was that with one of the lowest sequence identity with the target protein among the S100 family in the apo state. Based on the previously solved zebrafish holo-S100Z, a putative human holo-S100Z structure has been then calculated through homology modeling; the differences between the experimental human apo and calculated holo structure have been compared to those existing for other members of the family.

[The expression and antibody preparation of S100A10 protein].

OBJECTIVE: To prepare S100A10 protein and its specific polyclonal antibody. METHODS: The full-length gene fragment of S100A10 was amplified by PCR, and then cloned into pET28a(+) prokaryotic expression vector. After transformation, the vector was induced to express the recombinant (S100A10)(2) protein by IPTG in E.coli BL21 (DE3). The recombinant (S100A10)(2) was then purified by Ni-NTA resin. (S100A10)(2)-specific polyclonal antibody was prepared using the purified recombinant (S100A10)(2) protein as antigen to inoculate rabbit intradermally. The title and specificity of the polyclonal antibody were determined by ELISA and Western blotting. RESULTS: The study successfully constructed the prokaryotic recombinant expression vector pET28a(+)-(S100A10)(2), and obtained the purified recombinant (S100A10)(2) protein and polyclonal antibody with high titer and specificity. CONCLUSION: The prokaryotic expression and purification system for S100A10 has been established and polyclonal antibody of (S100A10)(2) been prepared, which provides helpful fools for further researches on S100A10.

[The antibodies to neurohormonal anti-genes as a criterion of early diagnostic and neurointoxication in workers of chemical enterprises].

The examination was applied to enterprise workers laboring in conditions of vinyl chloride (79 patients), caustic soda (24 patients) and 10 patients with professional chronic mercury intoxication. The differences are established concerning manifestation of autoimmune reactions of personnel working in conditions of chronic effecting of vinyl chloride distinct of parameters characterizing autoimmune reactions of personnel working under impact of another neuro-toxicants (vapors of metallic mercury). The increasing of auto-antibodies to MAG was detected in healthy personnel and increasing of concentrations of auto-antibodies to protein S-100 and DNA was detected in personnel with initial manifestations of neuro-intoxication. These occurrences testify availability, of different mechanisms underlying formation of neurological disorders. The study data confirms involvement of auto-antibodies to neuronal antigens into derangement of neural activity in personnel working in conditions of effect of vinyl chloride and vapors of metallic mercury. Hence, the new possibilities are opened in studying pathogenesis of occupational neuro-intoxications. The detection of auto-antibodies to proteins of neural tissue can be recommended as a criterion of early identification of damage of neural system in personnel working in conditions of chemical industry.

Immunohistochemical evaluation of neuroreceptors in healthy and pathological temporo-mandibular joint.

AIM: A study was performed on the articular disk and periarticular tissues of the temporo-mandibular joint (TMJ) with immunohistochemical techniques to give evidence to the presence of neuroreceptors (NRec) in these sites. METHODS: The study was carried out on tissue samples obtained from 10 subjects without TMJ disease and from 7 patients with severe TMJ arthritis and arthrosis. We use antibodies directed against following antigens: Gliofibrillary Acidic Protein (GFAP), Leu-7, Myelin Basic Protein (MBP), Neurofilaments 68 kD (NF), Neuron Specific Enolase (NSE), S-100 protein (S-100) and Synaptophysin (SYN). RESULTS: This study revealed that Ruffini's-like, Pacini's-like and Golgi's-like receptors can be demonstrated in TMJ periarticular tissues and that free nervous endings are present in the subsynovial tissues but not within the articular disk. We observed elongated cytoplamic processes of chondrocytes that demonstrated strong S-100 immunoreactivity but they were unreactive with all other antibodies. These cytoplamic processes were more abundant and thicker in the samples obtained from patients with disease TMJ. CONCLUSION: The results of this study confirm that different Nrec are detectable in TMJ periarticular tissues but they are absent within the articular disk. In the latter site, only condrocytic processes are evident, especially in diseased TMJ, and they might have been confused with nervous endings in previous morphological studies. Nevertheless the absence of immunoreactivity for NF, NSE and SYN proves that they are not of neural origin.

Purification and characterization of the human cysteine-rich S100A3 protein and its pseudo citrullinated forms expressed in insect cells.

High quantity and quality of recombinant Ca(2+)-binding proteins are required to study their molecular interactions, self-assembly, posttranslational modifications, and biological activities to elucidate Ca(2+)-dependent cellular signaling pathways. S100A3 is a unique member of the S100 protein family with the highest cysteine content (10%). This protein, derived from human hair follicles and cuticles, is characterized by an N-terminal acetyl group and irreversible posttranslational citrullination by peptidylarginine deiminase causing its homotetramer assembly. Insect cells, capable of introducing eukaryotic N-terminus and disulfide bonds, are an appropriate host in which to express this cysteine-rich protein. Four out of ten cysteines in the recombinant S100A3 form two intramolecular disulfide bridges that modulate its Ca(2+)-affinity. Three free thiol groups located at the C-terminus are predicted to form the high-affinity Zn(2+)-binding site. Citrullination of specific arginine residues in native S100A3 can be mimicked by site-directed mutagenic substitution of Arg/Ala. This chapter details our procedures used for the purification and characterization of the human S100A3 protein and its pseudo citrullinated forms expressed in insect cells.

X-ray structural analysis of S100 proteins.

X-ray crystallography is a potent and meanwhile fast technique to obtain detailed structural information of S100 proteins in their apo or metal ion-loaded state. S100 proteins crystallize in the absence or presence of Ca(2+) and Zn(2+) and the obtained crystals often diffract to high resolution yielding information on the ion-binding sites, conformation, and target interaction sites of the proteins. Here, I describe a general scheme to isolate and crystallize S100 proteins and the analysis of protein crystals using a modern synchrotron source.

Detection of Ca2+-binding S100 proteins in human saliva by HPLC-ESI-MS.

High-performance liquid chromatography (HPLC) coupled with electrospray ionization (ESI) mass -spectrometry (MS) is a relevant technique for the detection and relative quantitation of naturally occurring peptides and proteins. The peptide/protein mass is determined by deconvolution of the ESI-MS spectrum, and the resolution can be better than 1:10,000 with the instruments currently available. Accurate mass measurement, coupled with sufficient resolution, makes it possible to greatly restrict the enormous number of possible molecular formulas that might be represented by a specific molecular mass. As soon as the protein mass has been unequivocally attributed to a specific structure by means of different enzymatic and chemical treatments, the m/z values detected in the ESI spectrum can be utilized to reveal the protein and to perform its relative quantitation, by the extracted ion current (XIC) procedure, in an unlimited number of samples. This chapter describes the HPLC-ESI-MS experimental conditions which allow detecting and quantifying-in human saliva-different S100 proteins and their isoforms.

Analysis of S100 oligomers and amyloids.

The S100 proteins are a large family of 10-12 kDa EF-hand signaling proteins that bind calcium, and in some cases zinc and copper, functioning as central regulators in a diversity of cellular processes. These proteins have tissue, cell, and subcellular-specific expression patterns, and many have an extracellular function. Altogether, these properties underlie their functional diversity and involvement in several pathological conditions including cancer, inflammation, and neurodegeneration. S100 proteins exhibit considerable structural plasticity, being able to exist as monomers or assemble into dimers, higher oligomers, and amyloids, frequently in a metal-dependent manner. Many of these oligomers are functionally relevant, and S100 amyloids have been recently found in prostatic inclusions. Here, we report experimental procedures for the isolation and quantitation of S100 oligomers from tissues, purification of recombinant human S100 protein for assays and use as standards, and an amyloidogenesis assay that allows monitoring the formation of S100 ß-oligomers and amyloids in apo- and metal-bound S100 proteins.

Expression and purification of bioactive high-purity human S100A6 in Escherichia coli.

S100A6, as a member of S100 protein family, have biological functions in cell proliferation, differentiation, morphology, cytoskeletal organization and apoptosis. In the last three decades, S100A6 has been caught more and more attention. Here, we introduced a simple and efficient method for producing high-purity recombinant human S100A6 from Escherichia coli culture with low level of endotoxin. We further demonstrated its biological activities for triggering SH-SY5Y cells apoptosis in vitro. These results can facilitate the study of physiological and pathological roles of S100A6 and other members of S100 family proteins.

Tumor esofágico de células granulares: descripción de un tumor benigno poco frecuente / Granular cell tumor of the esophagus: description of an infrequent benign tumor

ResumenEl tumor de células granulares (TCG) es un tumor infrecuente descrito en 1926 por Abrikossoff. El 6% de estos tumores se encuentran en el tracto gastrointestinal siendo el esófago el lugar de mayor incidencia. El comportamiento suele ser benigno por lo que la mayoría de los TCG esofágicos son asintomáticos descubriéndose de forma casual en la realización de una gastroscopia. En el examen endoscópico aparece como una lesión polipoide o sésil, de aspecto submucoso, bien delimitada, recubierta de mucosa normal de color amarillento y con un aumento de su consistencia. La ecoendoscopia ha mejorado notablemente la evaluación de estas lesiones siendo actualmente la mucosectomía endoscópica el tratamiento de elección con una baja tasa de complicaciones. El estudio histológico de la pieza de resección evidencia características propias como la positividad para la proteína S-100. Presentamos 2 nuevos casos de TCG en esófago recientemente diagnosticados y se comentan los aspectos más relevantes de esta infrecuente enfermedad (AU)

Granular cell tumors (GCT) are infrequent tumors first described by Abrikossoff in 1926. Gastrointestinal involvement occurs in about 6% of GCT, the esophagus being the most frequent location. These tumors are usually benign and asymptomatic and are usually found incidentally when an upper gastrointestinal endoscopy is carried out for another reason, showing an isolated polyp or sessile submucosal nodule, covered by intact yellowish mucosa and with firm consistency. Endoscopic ultrasonography has significantly improved the diagnosis of these lesions. Nowadays endoscopic mucosectomy is the treatment of choice of esophageal GCT with a low frequency of complications. Histologic analysis of the surgical specimen shows specific characteristics such as positivity for S-100 protein. We present two new cases of esophageal GCT that were diagnosed recently and discuss the most relevant features of this infrequent disease (AU)

Refined crystal structures of human Ca(2+)/Zn(2+)-binding S100A3 protein characterized by two disulfide bridges.

S100A3, a member of the EF-hand-type Ca(2+)-binding S100 protein family, is unique in its exceptionally high cysteine content and Zn(2+) affinity. We produced human S100A3 protein and its mutants in insect cells using a baculovirus expression system. The purified wild-type S100A3 and the pseudo-citrullinated form (R51A) were crystallized with ammonium sulfate in N,N-bis(2-hydroxyethyl)glycine buffer and, specifically for postrefolding treatment, with Ca(2+)/Zn(2+) supplementation. We identified two previously undocumented disulfide bridges in the crystal structure of properly folded S100A3: one disulfide bridge is between Cys30 in the N-terminal pseudo-EF-hand and Cys68 in the C-terminal EF-hand (SS1), and another disulfide bridge attaches Cys99 in the C-terminal coil structure to Cys81 in helix IV (SS2). Mutational disruption of SS1 (C30A+C68A) abolished the Ca(2+) binding property of S100A3 and retarded the citrullination of Arg51 by peptidylarginine deiminase type III (PAD3), while SS2 disruption inversely increased both Ca(2+) affinity and PAD3 reactivity in vitro. Similar backbone structures of wild type, R51A, and C30A+C68A indicated that neither Arg51 conversion by PAD3 nor SS1 alters the overall dimer conformation. Comparative inspection of atomic coordinates refined to 2.15-1.40 Å resolution shows that SS1 renders the C-terminal classical Ca(2+)-binding loop flexible, which are essential for its Ca(2+) binding properties, whereas SS2 structurally shelters Arg51 in the metal-free form. We propose a model of the tetrahedral coordination of a Zn(2+) by (Cys)(3)His residues that is compatible with SS2 formation in S100A3.

Homogeneous immunosubtraction integrated with sample preparation enabled by a microfluidic format.

Immunosubtraction is a powerful and resource-intensive laboratory medicine assay that reports both protein mobility and binding specificity. To expedite and automate this electrophoretic assay, we report on advances to the electrophoretic immunosubtraction assay by introducing a homogeneous, not heterogeneous, format with integrated sample preparation. To accomplish homogeneous immunosubtraction, a step-decrease in separation matrix pore-size at the head of a polyacrylamide gel electrophoresis (PAGE) separation channel enables "subtraction" of target analyte when capture antibody is present (as the large immune-complex is excluded from PAGE), but no subtraction when capture antibody is absent. Inclusion of sample preparation functionality via small pore size polyacrylamide membranes is also key to automated operation (i.e., sample enrichment, fluorescence sample labeling, and mixing of sample with free capture antibody). Homogeneous sample preparation and assay operation allows on-the-fly, integrated subtraction of one to multiple protein targets and reuse of each device. Optimization of the assay is detailed which allowed for ~95% subtraction of target with 20% non-specific extraction of large species at the optimal antibody-antigen ratio, providing conditions needed for selective target identification. We demonstrate the assay on putative markers of injury and inflammation in cerebrospinal fluid (CSF), an emerging area of diagnostics research, by rapidly reporting protein mobility and binding specificity within the sample matrix. We simultaneously detect S100B and C-reactive protein, suspected biomarkers for traumatic brain injury (TBI), in ~2 min. Lastly, we demonstrate S100B detection (65 nM) in raw human CSF with an estimated lower limit of detection of 3.25 nM, within the clinically relevant concentration range for detecting TBI in CSF. Beyond the novel CSF assay introduced here, a fully automated immunosubtraction assay would impact a spectrum of routine but labor and time-intensive laboratory medicine assays.

Melanoma rectal: ¿Cuál es el tratamiento correcto? / Anorectal melanoma. What is the correct treatment?

El melanoma maligno primario de localización anorectal es una neoplasia poco frecuente y con una presentación atípica. Los autores presentan un caso y realizan un exhaustivo análisis sobre las consideraciones de su tratamiento quirúrgico (AU)

The primary anorectal malignant melanoma is an infrequent neoplasia with an atypical presentation. The authors have a case presentation and an exhaustive analysis and consideration over to surgical therapy (AU)

Expression, purification and fluorine-18 radiolabeling of recombinant S100A4: a potential probe for molecular imaging of receptor for advanced glycation endproducts in vivo?

Data concerning the pathophysiological role of extracellular S100A4, a member of the multigenic family of Ca(2+)-modulated S100 proteins, and its interaction with the receptor for advanced glycation endproducts (RAGE) or other putative receptors in tumorigenesis, metastasis, and inflammatory processes in vivo are scarce. One reason is the shortage of suitable radiotracer methods. We report a novel methodology using recombinant human S100A4 as potential probe for molecular imaging and functional characterization of this interaction. Therefore, human S100A4 was cloned as GST fusion protein in the bacterial expression vector pGEX-6P-1 and expressed in E. coli strain BL21. Purified recombinant human S100A4 was radiolabeled with the positron emitter fluorine-18 ((18)F) by conjugation with N-succinimidyl-4-[(18)F]fluorobenzoate ([(18)F]SFB). The radioligand [(18)F]fluorobenzoyl-S100A4 ((18)F-S100A4) was used in cell binding experiments in RAGE-bearing human melanoma cells and endothelial cells in vitro, and in both biodistribution experiments and small animal positron emission tomography (PET) studies in normal rats in vivo. The cellular association and tissue-specific distribution of (18)F-S100A4 in vitro and in vivo correlated well with the protein expression and anatomical localization of RAGE, e.g., in the vascular system and in lung. Compared to other S100 RAGE radioligands, the overall findings of this study indicate that extracellular S100A4 in vivo shows only a moderate interaction with RAGE and, furthermore, exhibits a substantially faster metabolic degradation. On the other hand, the approach allows the use of quantitative small animal PET and provides a novel probe to both delineate functional expression and differentiate multiligand interaction of RAGE under normal and pathophysiological conditions in rodent models of disease.

Purification and partial characterization of canine S100A12.

Canine S100A12 (cS100A12) is a calcium-binding protein of the S100 superfamily of EF-hand proteins, and its expression is restricted to neutrophils and monocytes. Interaction of S100A12 with the receptor for advanced glycation end products (RAGE) has been suggested to play a central role in inflammation. Moreover, S100A12 has been shown to represent a sensitive and specific marker for gastrointestinal inflammation in humans. Only human, porcine, bovine, and rabbit S100A12 have been purified to date, and an immunoassay for the quantification of S100A12 is available only for humans. Therefore, the aim of this study was to develop a protocol for the purification of S100A12 and to partially characterize this protein in the dog (Canis lupus familiaris) as a prelude to the development of an immunologic method for its detection and quantification in canine serum and fecal specimens. Leukocytes were isolated from canine whole blood by dextran sedimentation, and canine S100A12 was extracted from the cytosol fraction of these cells. Further purification of cS100A12 comprised of ammonium sulfate precipitation, hydrophobic interaction chromatography, and strong cation- and anion-exchange column chromatography. Canine S100A12 was successfully purified from canine whole blood. The relative molecular mass of the protein was estimated at 10,379.5 and isoelectric focusing revealed an isoelectric point of 6.0. The approximate specific absorbance of cS100A12 at 280 nm was determined to be 1.78 for a 1 mg/ml solution. The N-terminal AA sequence of the first 15 residues of cS100A12 was Thr-Lys-Leu-Glu-Asp-His-X-Glu-Gly-Ile-Val-Asp-Val-Phe-His, and revealed 100% identity with the predicted protein sequence available through the canine genome project. Sequence homology for the 14 N-terminal residues identified for cS100A12 with those of feline, bovine, porcine, and human S100A12 was 78.6%. We conclude that canine S100A12 can be successfully purified from canine whole blood using the described methods.

Purification of antimicrobial peptides from human skin.

Human skin is a rich source of human antimicrobial peptides. Its cellular source is the keratinocyte, which terminally differentiates in the uppermost parts of the skin, eventually forming the stratum corneum, the horny layer. The easy availability of human stratum corneum makes it possible to identify and characterize human antimicrobial peptides with a biochemical approach. Moreover, the availability of lesional scales of patients with psoriasis, an inflammatory skin disease, allows the identification of human-inducible peptide antibiotics, which are absent in healthy skin. With this strategy, the beta-defensins hBD-2 and hBD-3, RNase-7 as well as psoriasin/S100A7 have been discovered as human antimicrobial peptides and proteins. A detailed description of the strategies and methods is presented, which allowed a successful identification and characterization of human antimicrobial peptides. We used various HPLC techniques, combined with antimicrobial testing as read-out system. In parallel, SDS-PAGE analyses as well as electrospray ionization mass spectrometry were used for further biochemical characterization as well as purity assessment.

Design of high-affinity S100-target hybrid proteins.

S100B and S100A10 are dimeric, EF-hand proteins. S100B undergoes a calcium-dependent conformational change allowing it to interact with a short contiguous sequence from the actin-capping protein CapZ (TRTK12). S100A10 does not bind calcium but is able to recruit the N-terminus of annexin A2 important for membrane fusion events, and to form larger multiprotein complexes such as that with the cation channel proteins TRPV5/6. In this work, we have designed, expressed, purified, and characterized two S100-target peptide hybrid proteins comprised of S100A10 and S100B linked in tandem to annexin A2 (residues 1-15) and CapZ (TRTK12), respectively. Different protease cleavage sites (tobacco etch virus, PreScission) were incorporated into the linkers of the hybrid proteins. In situ proteolytic cleavage monitored by (1)H-(15)N HSQC spectra showed the linker did not perturb the structures of the S100A10-annexin A2 or S100B-TRTK12 complexes. Furthermore, the analysis of the chemical shift assignments ((1)H, (15)N, and (13)C) showed that residues T102-S108 of annexin A2 formed a well-defined alpha-helix in the S100A10 hybrid while the TRTK12 region was unstructured at the N-terminus with a single turn of alpha-helix from D108-K111 in the S100B hybrid protein. The two S100 hybrid proteins provide a simple yet extremely efficient method for obtaining high yields of intact S100 target peptides. Since cleavage of the S100 hybrid protein is not necessary for structural characterization, this approach may be useful as a scaffold for larger S100 complexes.