Super-ego in patients with coronary artery disease

In this paper, we explored the super-ego ofpatients with coronary artery disease. Research results have confirmed the initial hypothesis that a significant number of patients with coronary artery disease has rigorous super-ego. Among patients with coronary artery disease (N=50), and control group (N=50), we have found significant differences in the quality of super-ego and ego attitude towards the demands of the super-ego. The results of this research contribute to understanding the impact of psychological factors in coronary artery disease.     

AXIN-1 protein expression and localization in glioblastoma

The etiology and pathogenesis of tumors of the central nervous system are still inadequately explained. In the present study the expression patterns of a critical molecular component of wnt signaling pathway - axin I was investigated in 42 patients with glioblastoma, the most aggressive form of glial tumors. Immunostaining and image analysis revealed the quantity and localization of the protein. Downregulation of this tumor suppressor expression was observed in 31% of tumors when compared to the levels of axin in healthy brain tissues. Axin was observed in the cytoplasm in 69% of glioblastoma samples, in 21.4% in both the cytoplasm and nucleus and 9.5% had expression solely in the nucleus. Mean values of relative axin's expression obtained by image analysis showed that the highest relative quantity of axin was measured when the protein was in the nucleus and the lowest relative quantity of axin when the protein was localized in the cytoplasm. Investigation on axin's existence at the subcellular level in glioblastomas suggests that axin's expression and spatial regulation is a dynamic process. Despite increasing knowledge on glioma biology and genetics, the prognostic tools for glioblastoma still need improvement. Our findings on expression of axin 1 may contribute to better understanding of glioblastoma molecular profile.     

Chondromyxoid fibroma of the second metacarpal bone--a case report

This report describes a chondromyxoid fibroma of the second metacarpal bone in a 32-year-old female patient. Chondromyxoid fibroma is a rare, benign, slow-growing bone tumor of cartilaginous origin. Tumor has a high recurrance rate. Our aim was to show successful treatment of a metacarpal chondromyxoid fibroma with wide resection and implantation of finger join endoprosthesis.     

Enzyme kinetics and the maximum entropy production principle

A general proof is derived that entropy production can be maximized with respect to rate constants in any enzymatic transition. This result is used to test the assumption that biological evolution of enzyme is accompanied with an increase of entropy production in its internal transitions and that such increase can serve to quantify the progress of enzyme evolution. The state of maximum entropy production would correspond to fully evolved enzyme. As an example the internal transition ES?EP in a generalized reversible Michaelis-Menten three state scheme is analyzed. A good agreement is found among experimentally determined values of the forward rate constant in internal transitions ES→EP for three types of β-Lactamase enzymes and their optimal values predicted by the maximum entropy production principle, which agrees with earlier observations that β-Lactamase enzymes are nearly fully evolved. The optimization of rate constants as the consequence of basic physical principle, which is the subject of this paper, is a completely different concept from a) net metabolic flux maximization or b) entropy production minimization (in the static head state), both also proposed to be tightly connected to biological evolution.     

Immunocapture-Selected Reaction Monitoring Screening Facilitates the Development of ELISA for the Measurement of Native TEX101 in Biological Fluids

Monoclonal antibodies that bind the native conformation of proteins are indispensable reagents for the development of immunoassays, production of therapeutic antibodies and delineating protein interaction networks by affinity purification-mass spectrometry. Antibodies generated against short peptides, protein fragments, or even full length recombinant proteins may not bind the native protein form in biological fluids, thus limiting their utility. Here, we report the application of immunocapture coupled with selected reaction monitoring measurements (immunocapture-SRM), in the rapid screening of hybridoma culture supernatants for monoclonal antibodies that bind the native protein conformation. We produced mouse monoclonal antibodies, which detect in human serum or seminal plasma the native form of the human testis-expressed sequence 101 (TEX101) protein—a recently proposed biomarker of male infertility. Pairing of two monoclonal antibodies against unique TEX101 epitopes led to the development of an ELISA for the measurement of TEX101 in seminal plasma (limit of detection: 20 pg/ml) and serum (limit of detection: 40 pg/ml). Measurements of matched seminal plasma samples, obtained from men pre- and post-vasectomy, confirmed the absolute diagnostic specificity and sensitivity of TEX101 for noninvasive identification of physical obstructions in the male reproductive tract. Measurement of male and female serum samples revealed undetectable levels of TEX101 in the systemic circulation of healthy individuals. Immunocapture-SRM screening may facilitate development of monoclonal antibodies and immunoassays against native forms of challenging protein targets.Monoclonal antibodies that bind the native form of a protein are indispensable for the development of sensitive immunoassays, production of therapeutic antibodies and for studying protein interaction networks by affinity purification-mass spectrometry (1, 2). Large-scale purification of native proteins from biological samples may be challenging, so recombinant proteins or protein fragments are often used for antibody production. Antibodies produced against short peptides, protein fragments, or even full length recombinant proteins, however, may not bind the native protein conformation present in biological fluids, thus limiting the utility of antibodies. Rapid screening of antibody-producing hybridoma clones for native protein binders requires highly specific and sensitive assays, performed under nondenaturing conditions. Here, we report the capability of an immunocapture-SRM assay to facilitate fast screening of hybridoma cultures for monoclonal antibodies that recognize the native conformation of testis-expressed sequence 101 (TEX101)¹ protein in biological fluids.Recently, we discovered, verified, and validated two proteins, testis-specific protein TEX101 and epididymis-specific protein ECM1, as biomarkers for the differential diagnosis of azoospermia (3, 4). Combination of TEX101 and ECM1 proteins measured in seminal plasma could differentiate between normal spermatogenesis, obstructive azoospermia (OA), and nonobstructive azoospermia (NOA) with very high diagnostic sensitivity and specificity. TEX101 levels in seminal plasma also facilitated classification of NOA subtypes of hypospermatogenesis, maturation arrest and Sertoli cell-only syndrome (5). A clinical laboratory test for TEX101 in seminal plasma may confirm the success of vasectomy or vasovasostomy, eliminate diagnostic testicular biopsies, and predict the success of sperm cell retrieval for assisted reproduction.Human TEX101 is a membrane GPI-anchored protein encoded by the TEX101 gene, located in the 19q13.31 region of chromosome 19. According to the Human Protein Atlas, TEX101 expression is restricted to testicular tissue and male germ cells, with no evidence of expression in any other human tissue or cell type (6). Investigation of the function of mouse TEX101 demonstrated its direct role in fertilization (7–9).We initially measured TEX101 levels in seminal plasma by mass spectrometry-based selected reaction monitoring (SRM) and immuno-SRM assays, with limits of detection of 120 and 5 ng/ml, respectively (4, 5). However, because of the ultra-wide range of TEX101 concentrations in seminal plasma of infertile and healthy men (0.5 ng/ml to 50,000 ng/ml) and theoretically zero levels for some azoospermic patients, a sensitive TEX101 immunoassay is required to develop a clinical laboratory test. In addition to immunoassay, monoclonal antibodies against native TEX101 would allow investigating its interactome and revealing its functional role in spermatogenesis and male fertility. Because TEX101 may emerge as a novel biomarker of male infertility, in this work we focused on the development of an ELISA for sensitive measurement of TEX101 in seminal plasma and serum.Our initial efforts to develop a TEX101 immunoassay using commercially available polyclonal antibodies were not successful. We found that commercial antibodies recognized only the denatured form of TEX101 and were useful for immunohistochemistry and Western blots, but not for the analysis of native TEX101 in seminal plasma. Here, we describe the production of mouse monoclonal antibodies against native TEX101, screening of antibody-producing clones by the two-step immunocapture and SRM assay, development of a sensitive ELISA and measurement of TEX101 in seminal plasma and serum (Fig. 1).Open in a separate windowFig. 1.Pipeline for the production of mouse monoclonal anti-TEX101 antibodies and screening of colonies using two-step immunocapture-SRM assay. Screening included the coating of microtiter plates with sheep anti-mouse IgG antibodies, the addition of hybridoma cell supernatants, incubation with seminal plasma containing the native form of TEX101 followed by trypsin digestion and SRM analysis. Two-step immunocapture followed by SRM detection facilitated rapid screening of antibody-producing colonies and provided the following advantages: no requirement for previously developed TEX101 antibodies, small scale antibody production on 96-well plates, screening of low amounts of the newly-produced antibodies and direct selection of antibodies against the native form of TEX101. Eventually, all positive clones were expanded and a sensitive immunofluorescent assay for TEX101 was developed in seminal plasma and serum.     

Pentadecapeptide BPC 157 Reduces Bleeding and Thrombocytopenia after Amputation in Rats Treated with Heparin,Warfarin, L-NAME and L-Arginine

Background

BPC 157 is a stable gastric pentadecapeptide recently implicated with a role in hemostasis. While NO is largely implicated in hemostatic mechanisms, in tail-amputation-models under heparin- and warfarin-administration, both the NO-synthase (NOS)-blocker, L-NAME (prothrombotic) and the NOS-substrate L-arginine (antithrombotic), were little investigated. Objective. To investigate the effect of L-NAME and L-arginine on hemostatic parameters, and to reveal the effects of BPC 157 on the L-NAME- and L-arginine-induced hemostatic actions under different pathological condition: tail amputation without or with anticoagulants, heparin or warfarin.

Methods

Tail amputation, and/or i.v.-heparin (10 mg/kg), i.g.-warfarin (1.5 mg/kg/day for 3 days) were used in rats. Treatment includes BPC 157, L-NAME, L-arginine, per se and their combination.

Results

After (tail) amputation, with or without i.v.-heparin or i.g.-warfarin, BPC 157 (10 μg/kg, 10 ng/kg, i.p., i.v. (heparin), 10 μg/kg i.g. (warfarin)) always reduced bleeding time and/or haemorrhage and counteracted thrombocytopenia. As for L-NAME and/or L-arginine, we noted: L-arginine (100 mg/kg i.p.)–rats: more bleeding, less/no thrombocytopenia; L-NAME (5 mg/kg i.p.)-rats: less bleeding (amputation only), but present thrombocytopenia; L-NAME+L-arginine-rats also exhibited thrombocytopenia: L-NAME counteracted L-arginine-increased bleeding, L-arginine did not counteract L-NAME-thrombocytopenia. All animals receiving BPC 157 in addition (BPC 157μg+L-NAME; BPC 157μg+L-arginine, BPC 157μg+L-NAME+L-arginine), exhibited decreased haemorrhage and markedly counteracted thrombocytopenia.

Conclusions

L-NAME (thrombocytopenia), L-arginine (increased haemorrhage) counteraction and BPC 157 (decreased haemorrhage, counteracted thrombocytopenia) with rescue against two different anticoagulants, implicate a BPC 157 modulatory and balancing role with rescued NO-hemostatic mechanisms.     

Changes in cell surface proteins during differentiation of mouse embryonal carcinoma cells

The cell surface proteins of teratocarcinoma-derived embryonal carcinoma cells (ECC), of parietal endoderm (Pys-2 and F9-AC cl 9), and of fibroblasts (OTT6050f) were radioiodinated by a lactoperoxidase method and analyzed by two-dimensional gel electrophoresis. The combined electrophoretic profiles of proteins from a number of ECC lines allowed the determination of eight ECC-unique polypeptides. Parietal endoderm and fibroblast expressed their own unique polypeptides. The two parietal endoderm-specific polypeptides are identical to the subunits of laminin. Retinoic acid-induced differentiation of one ECC line (F9) resulted in the disappearance of polypeptides specific for ECC and the appearance of those specific for the parietal endoderm.     

Brain and sperm cell surface antigen (NS-4) on preimplantation mouse embryos

Antiserum prepared in rabbit against 4-day-old mouse cerebellum (anti-NS-4 serum) reacts in the complement-mediated cytotoxicity test with unfertilized and fertilized mouse eggs, cleavage stage embryos, and cells of the trophoblast and inner cell mass of the mouse blastocyst. This activity is specifically removed by absorption of antiserum with adult mouse brain and epididymal sperm but not with adult liver, spleen, kidney, and thymocytes. The antiserum reacts most strongly with cells of the trophoblast and inner cell mass and, in order of decreasing reactivity, with four- to eight-cell stage embryos, zygotes, unfertilized eggs, and two-cell stage embryos.     

Influence of different etching times on dentin surface morphology

The aim of this study is to investigate the influence of different etching times on demineralized dentin surface morphology using scanning electron microscopy and qualitative line microanalysis of chemical structure. Two sample groups, consisting of 30 first premolar teeth in each group, were established. Teeth were cut at the half-distance between the enamel-dentin junction and the pulp. The first group of specimens was etched for 10 seconds and the second group for 30 seconds. 37% ortophosphoric acid was used. SEM (scanning electron microscopy) was utilized to observe the following parameters: number and diameter of dentinal tubules, dentinal and intertubular dentinal surface percentage, appearance of the dentin surface porous zone containing smear layer and demineralized residual collagen particles with dentin demineralization products in acid globules, and dissolved peritubular dentin cuff. After calculating measurements of central tendency (X,C, Mo, SD), Kolmogorov-Smirnov and Student t-test were performed to confirm the quantitative results, and the chi2-test was run to produce qualitative data. In contrast to the 10-second etching time, the increased etching time of 30 seconds resulted in the following findings: (1) an increased number of dentinal tubules (p < 0.05), (2) an increase in dentinal tubule diameter (p < 0.05), (3) an increase in dentinal tubule surface percentage (p < 0.001), (4) a decrease in intertubular dentinal surface percentage (p < 0.001), (5) appearance of dentin surface porous zone containing smear layer and demineralized residual collagen particles with dentin demineralization products in acid globules (p < 0.001), and (6) completely dissolved peritubular dentin cuff (p <0.001). Therefore, different etching times using the same phosphoric acid concentration result in different morphological changes in demineralized dentin surface. Moreover, based on a comparison with current studies, prolonged etching time causes morphological changes to dentin surface. Such changes, have, in turn, negative effects on the dentin hybridization process.