Characterization of proteins from boar prostate

PROBLEM: Most components of seminal plasma are secreted by accessory sexual glands: seminal vesicle, prostate gland and bulbourethral gland. The portion of proteins secreted by prostate gland differs in various species. Characterization of boar prostate proteins is the subject of this communication. METHODS OF STUDY: Proteins of boar prostate gland were separated by affinity chromatography on heparin-polyacrylamide to non-heparin-binding (H-) and heparin-binding (H+) fractions. The H- and H+ fractions were subjected to reverse phase high performance liquid chromatography (RP HPLC) and their elution profiles were compared with those of the H- and H+ fractions of boar seminal plasma. The isolated proteins were characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), immunodetection, N-terminal amino acid sequencing and mass spectrometry (MALDI). RESULTS: The following proteins of boar prostate secretion were identified: beta-microseminoprotein, serotransferrin, serum albumin, myoglobin and PSP I and PSP II spermadhesins. CONCLUSION: Presented results demonstrate composition of the main proteins of boar prostate secretion. Beta-Microseminoprotein was found to be a major protein of prostate secretion. PSP I and PSP II, major proteins of the H- fraction of boar seminal plasma, were found in boar prostate secretion in lower amounts. The major proteins of the H+ fraction of boar seminal plasma (AQN, AWN) were not detected in prostate secretion.     

Anti-cyclic citrullinated peptide antibodies in patients with juvenile idiopathic arthritis

We analysed the presence of anti-cyclic citrullinated peptide (anti-CCP) and anti-keratin (AKA) antibodies of the IgG class in sera of patients with defined juvenile idiopathic arthritis (JIA) of various subgroups with more than one year duration of the disease. Enzyme-linked immunosorbent assay (Immunoscan RA, Eurodiagnostica, The Netherlands) and an indirect immunofluorescence (IIF) test on rat oesophagus substrate (ImmuGloTM, Immco Diagnostics, Buffalo, USA) were used for the detection and quantification of anti-CCP and AKA antibodies in 140 patients with JIA (64 male and 76 female) aged 2-47 years (median 16.5 years). Overall, anti-CCP were found in 7/140 (5.0%) patients including 3/52 RF negative polyarthritis, 2/18 RF positive polyarthritis, 1/15 enthesitis related arthritis and 1/5 unclassifiable arthritis. AKA were detected in 40/140 patients (28.6%, p = 0.04) including 2/11 systemic arthritis, 2/32 oligoarthritis, 18/52 patients with RF negative polyarthritis (34.6%, p = 0.01), 14/18 RF positive polyarthritis (77.8%, p = 0.000002), 2/15 enthesitis related arthritis and 2/3 psoriatic arthritis. While simultaneous negativity for AKA and anti-CCP occurred in most (97/140; 69.3%) studied cases, simultaneous antibody positivity was found only in few (4/140; 2.9%) studied samples. We conclude that while AKA measured using IIF on rat esophagus can be detected approximately in one third of patients with definite JIA with more than 1 year duration of the disease, only rare occurrence of anti-CCP was observed. We conclude that AKA seem to be partly useful to confirm JIA diagnosis, however, useless to follow-up severity or activity in JIA patients. Anti-CCP do not have any additional value in MA cohort in comparison to RA where their diagnostic and prognostic importance was reported.     

Creep Resistance of S304H Austenitic Steel Processed by High-Pressure Sliding

Sheets of coarse-grained S304H austenitic steel were processed by high-pressure sliding (HPS) at room temperature and a ultrafine-grained microstructure with a mean grain size of about 0.14 µm was prepared. The microstructure changes and creep behavior of coarse-grained and HPS-processed steel were investigated at 500–700 °C under the application of different loads. It was found that the processing of S304H steel led to a significant improvement in creep strength at 500 °C. However, a further increase in creep temperature to 600 °C and 700 °C led to the deterioration of creep behavior of HPS-processed steel. The microstructure results suggest that the creep behavior of HPS-processed steel is associated with the thermal stability of the SPD-processed microstructure. The recrystallization, grain growth, the coarsening of precipitates led to a reduction in creep strength of the HPS-processed state. It was also observed that in the HPS-processed microstructure the fast formation of σ-phase occurs. The σ-phase was already formed during slight grain coarsening at 600 °C and its formation was enhanced after recrystallization at 700 °C.     

Proprioceptive Neuromuscular Facilitation in Combination with Electrical Stimulation: Combined Treatment in Comparison to Each Treatment Alone

The objective of the study was a quantitative examination of Proprioceptive Neuromuscular Facilitation (PNF) exercise in simultaneous combination with FES of lower extremity muscles in comparison to voluntary movement, training with PNF alone, or training with FES alone. Two subjects were monitored during a one‐month rehabilitation period. The PNF pattern included flexion, adduction, and external rotation of the hip, knee flexion, and dorsiflexion with inversion of the ankle, a pattern similar to the swing phase of walking. Quantitative measurements were conducted by using goniometers on the hip, knee, and ankle joints. Major changes were found in the hip angle. Improvements in goniograms were greatest during the first week, smaller during the second week, and showed only a slight positive trend in the last two weeks. The measurements made two months after the start of training showed somewhat lower values in comparison to previous sessions.     

Cytoplasmic delivery and nuclear targeting of synthetic macromolecules

Delivery of macromolecular drugs (e.g. antisense oligonucleotides, polymer-drug conjugates, etc.) designed to work in specific sites inside cells is complicated as macromolecules typically have access to fewer biological compartments than small molecules. To better understand the fate of macromolecules in cells and begin to alter that fate, we investigated the internalization and subcellular fate of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers and HPMA copolymer-drug conjugates in Hep G2 and A2780 cells. The subcellular fate of fluorescently labeled polymers was monitored by confocal microscopy and subcellular fractionation. Initially, the HPMA copolymers and HPMA copolymer-drug conjugates were internalized by endocytosis and remained in endosomes/lysosomes. At longer incubation times (>8 h), small amounts of the HPMA copolymers were observed to enter the cytoplasm and accumulate in the nucleus of the cells. Nuclear accumulation was confirmed after cytoplasmic microinjection. Oligonucleotides conjugated via lysosomally degradable spacers entered into the cytoplasm and nucleus of the cells faster than the polymers. The effect of the subcellular location was correlated to the toxicity of the photosensitizer, mesochlorin e(6) (Mce(6))-HPMA copolymer conjugates. The plasma membrane and late endosomes were more sensitive to damage by Mce(6). Targeting the polymer conjugates to the nucleus with the nuclear localization sequence (NLS) as well as conjugating the Mce(6) via a degradable spacer increased cell adhesion and uptake, promoted their entry into the cytoplasm and nucleus of the cells, and increased their toxicity. To further promote entry of the polymers into the cytoplasm and nucleus of the cells, the protein transduction domain, Tat peptide, was conjugated to the HPMA copolymers. This resulted in high binding to the cell membrane, but also facilitated rapid (<5 min) entry of the macromolecules into the cytoplasm and nucleus of cells. These results will prove valuable in the future design of macromolecular therapeutics.     

Coupling of the antitumoral enzyme bovine seminal ribonuclease to polyethylene glycol chains increases its systemic efficacy in mice

Bovine seminal ribonuclease (BS-RNase) is an antitumoral active enzyme exhibiting specific antitumoral action against a number of different cancer cell lines. However, its systemic use is limited by its pharmacokinetic properties and antigenicity. Therefore, it was conjugated to polyethylene glycol (PEG) chains to overcome these problems. Measurement of aspermatogenic effects of the preparation after s.c. injection and injection into the scrotum was chosen as a model for the distribution of the enzyme in the body mediated by the linkage to PEG chains. Additionally, the antigenicity of BS-RNase coupled to PEG chains (BS-RNase-PEG) was compared to that of free BS-RNase, as antigenicity is known to be one of the main obstacles in the use of protein-based drugs. BS-RNase-PEG caused aspermatogenic effects after systemic administration to mice in very low concentrations at which free BS-RNase is not effective. Moreover, BS-RNase possessed a very low antigenicity as long as it was coupled to the PEG chains. In order to investigate the antitumoral efficacy of BS-RNase-PEG in vivo, preliminary experiments on the effect of the conjugate on neuroblastoma growth in mice were performed in a UKF-NB-3 xeno-transplantate model, demonstrating a drastically increased anti-tumoral activity of the conjugate compared to the free enzyme.     

Distinct Immunoregulatory Mechanisms in Mesenchymal Stem Cells: Role of the Cytokine Environment

Mesenchymal stem cells (MSCs) represent a population of cells which have the ability to regulate reactivity of T and B lymphocytes by multiple mechanisms. The immunoregulatory activities of MSCs are strictly influenced by the cytokine environment. Here we show that two functionally distinct cytokines, interleukin-4 (IL-4) and interferon-γ (IFN-γ), significantly potentiate the ability of MSCs to inhibit IL-10 production by activated regulatory B cells (Bregs). However, MSCs in the presence of IL-4 or IFN-γ inhibit the IL-10 production by different mechanisms. Preincubation of MSCs with IFN-γ led to the suppression, but pretreatment with IL-4 of neither MSCs nor B cells resulted in the suppression of IL-10 production. The search for candidate regulatory molecules expressed in cytokine-treated MSCs revealed different patterns of the gene expression. Pretreatment of MSCs with IFN-γ, but not with IL-4, induced expression of indoleamine-2,3-dioxygenase, cyclooxygenase-2 and programmed cell death-ligand 1. To identify the molecule(s) responsible for the suppression of IL-10 production, we used specific inhibitors of the putative regulatory molecules. We found that indomethacine, an inhibitor of cyclooxygenase-2 (Cox-2) activity, completely abrogated the inhibition of IL-10 production in cultures containing MSCs and IFN-γ, but had no effect on the suppression in cell cultures containing MSCs and IL-4. The results show that MSCs can inhibit the response of B cells to one stimulus by different mechanisms in dependence on the cytokine environment and thus support the idea of the complexity of immunoregulatory action of MSCs.     

The identification of small molecules that stimulate retinal pigment epithelial cells: potential novel therapeutic options for treating retinopathies

Introduction: Combinatory strategies using pharmacology and stem cell therapy have emerged due to their potential in the treatment of retinal pigment epithelium (RPE) cell related diseases, and a variety of different stem cell sources have been evaluated both in animal models and in humans. RPE cells derived from human embryonic stem cells (hESCs) and human induced pluripotent cells (hiPSCs) are already in clinical trials, holding great promise for the treatment of age-related macular disease (AMD) and hereditary RPE-related retinal dystrophies. Highly efficient protocol for RPE generations have been developed, but they are still time-consuming and laborious.

Areas covered: The authors review RPE related diseases, as well as the known functions of RPE cells in retinal homeostasis. The authors also discuss small molecules that target RPE in vivo as well as in vitro to aid RPE differentiation from pluripotent stem cells clinically. The authors base this review on literature searches performed through PubMed.

Expert opinion: Using high-throughput systems, technology will provide the possibility of identifying and optimizing molecules/drugs that could lead to faster and simpler protocols for RPE differentiation. This could be crucial in moving forward to create safer and more efficient RPE-based personalized therapies.