Culture of newborn monkey liver epithelial progenitor cells in chemical defined serum-free medium

Studies with hepatic progenitor cells from non-human primates would allow better understanding of their human counterparts. In this study, rhesus monkey liver epithelial progenitor cells (mLEPCs) were derived from a small piece of newborn livers in chemical defined serum-free medium. Digested hepatic cells were treated in Ca²⁺-containing medium to form cell aggregates. Two types of cell aggregates were generated: elongated spindle cells and polygonal epithelial cells. Elongated spindle cells were expressed as vimentin and brachyury, and they were disappeared within 5 d in our cultures. The remaining type consisted of small polygonal epithelial cells that expressed cytokeratin 7 (CK7), CK8, CK18, nestin, CD49f, and E-cad, the markers of hepatic stem cells, but were negative for α-fetoprotein, albumin, and CK19. They can proliferate and be passaged, if on laminin or rat tail collagen gel, to initiate colonies. When cultured with dexamethasone and oncostatin M, the expression of mature hepatocyte markers, such as α-1-antitrypsin, intracytoplasmic glycogen storage, indocyanine green uptake, and lipid droplet generation, were induced in differentiated cells. If transferred onto mouse embryonic fibroblasts feeders, they gave rise to CK19-positive cholangiocytes with formation of doughnut-like structure. Thus, mLEPCs with bipotency were derived from newborn monkey liver and may serve as a preclinical model for assessment of cell therapy in humans.     

New data on small mammals in the Kazakh Uplands

Small mammals were studied in the Kazakh Uplands in the spring and fall of 2008. The trapping studies revealed 10 species. Abundances of the animals were low in the four main distinct characteristic biotopes of Bayanaul National Park, but those of biotope dominants were high. In the Kazakh Uplands, rodents and insectivores are clearly restricted to certain biotopes. Biodiversity indices for small mammal communities are low, indicating that the community structure is disturbed.     

Tyrosinases of motile Azospirillum strains

A number of motile strains of Azospirillum brasilense, A. lipoferum, and A. irakense, were found to possess tyrosinase activity both on the surface of and inside the cells. A. brasilense Sp245, Sp7, and A. irakense KBC-1 each possessed two forms of tyrosinase of different molecular masses; A. lipoferum 43, A. lipoferum 59b, and A. irakense KA-3 each had a single tyrosinase form of approximately the same molecular mass; and A. brasilense Sp107 possessed a single form of tyrosinase different from all the other forms.     

Construction and Characterization of a CTLA-4-Targeted scFv–Melittin Fusion Protein as a Potential Immunosuppressive Agent for Organ Transplant

Immunotoxins with selective cytotoxicity are frequently used as therapeutic immunosuppressive agents in solid-organ transplantation because of their efficiency and high specificity. In this study, we present a new recombinant immunotoxin termed anti-CTLA-4-scFv–melittin prepared from Escherichia coli aimed at clearing activated T cells at the same time avoiding all-round decline in systematic immunity. This fusion protein is composed of anti-CTLA-4-scFv unit and melittin analog unit with properties of low immunogenicity and selective cytotoxicity to CTLA-4-positive T cells. In preliminary biological activity assays, our results confirmed the feasibility of activated T cell clearance strategy and there were significant differences in cell survival rates between CTLA-4-positive group and control group at all experimental concentrations of the immunotoxin. The selective cytotoxicity, low immunogenicity, and low production cost make it an attractive alternate to traditional immunosuppressants.     

U-Shape Suppressive Effect of Phenol Red on the Epileptiform Burst Activity via Activation of Estrogen Receptors in Primary Hippocampal Culture

Phenol red is widely used in cell culture as a pH indicator. Recently, it also has been reported to have estrogen-like bioactivity and be capable of promoting cell proliferation in different cell lines. However, the effect of phenol red on primary neuronal culture has never been investigated. By using patch clamp technique, we demonstrated that hippocampal pyramidal neurons cultured in neurobasal medium containing no phenol red had large depolarization-associated epileptiform bursting activities, which were rarely seen in neurons cultured in phenol red-containing medium. Further experiment data indicate that the suppressive effect of the phenol red on the abnormal epileptiform burst neuronal activities was U-shape dose related, with the most effective concentration at 28 µM. In addition, this concentration related inhibitory effect of phenol red on the epileptiform neuronal discharges was mimicked by 17-β-estradiol, an estrogen receptor agonist, and inhibited by ICI-182,780, an estrogen receptor antagonist. Our results suggest that estrogen receptor activation by phenol red in the culture medium prevents formation of abnormal, epileptiform burst activity. These studies highlight the importance of phenol red as estrogen receptor stimulator and cautions of careful use of phenol red in cell culture media.     

Insulin-Like Peptides of the Cerebropleural Ganglion of the Mollusc Anodonta cygnea: Isolation, Purification, and Radioligand Analysis

Cerebropleural ganglia from 4000 individuals of the mollusc Anodonta cygnea were submitted to procedures developed for isolation of vertebrate pancreatic insulins: homogenization and extraction, stage-like isoelectrical sedimentation, and ion-exchange chromatography. As a result of purification of the obtained preparation, using high-effective liquid chromatography, there were identified 7 protein peaks differing by time of retention on the reverse-phase sorbent in acetonitryl gradient and designated as insulin-related peptides (IRP), IRP1-IRP7. The material was characterized by the peptide ability to inhibit specific binding of ¹²⁵I-insulin and of insulin-related factor-1 (¹²⁵I-IGF-1) by plasma membranes of the rat liver and brain. The IC₅₀ value of peptide concentration (nM) able to replace 50% of the labeled hormone bound with the receptor amounted in the insulin radioreceptor system for IRP1 to 330, for IRP3 to 130, for IRP4 to 17, for IRP5 to130, for IRP6 to 420 nM. Peptide IRP7 at a maximal concentration (10⁴ ng/ml) replaced less than 50% of labeled hormone, whereas in IRP2 no inhibitory ability was detected under these experimental conditions. The IC₅₀ value in the case of ¹²⁵I-IGF-1 amounted for IRP1, IRP4, and IRP5 to17, for IRP2 to 50, for IRP3 to 83, for IRP6 to 133 nM. IRP7 at a concentration of 10⁴ ng/ml replaced less than 50% of labeled hormone. The same high relative affinity of the peptide IRP4 (12% of activity of standard insulin and IGF-1) to both receptor types is revealed. The results of analysis in two types of hormonal test systems indicate the ability of the insulin-related peptides of the anodonta cerebropleural ganglion to interact with the vertebrate receptor of insulin and IGF-1. This gives grounds to suggest the presence of the metabolic and growth-stimulating properties in these peptides. For the first time, the IGF-1 activity is revealed in insulin-like molecules in invertebrates. Taking into account the chromatographically revealed differences of physicochemical characteristics of individual IRP as well as predominance of their IGF-1-binding properties, there is suggested another organization of the IRP receptor-binding domains in IPR of this mollusc species, as compared with mammalian insulins.     

The Role of the Vascular Factor in the Reorganization of Water Metabolism in Denervated Liver after Bacterial Endotoxin Poisoning

Intoxication with bacterial lipopolysaccharide (endotoxin) is accompanied by considerable rearrangements in the systems of blood microcirculation and water metabolism of the liver. These rearrangements are manifested as increased sinusoid area, changed total area of the cytoplasm and nuclei as well as the nucleocytoplasmic ratio in hepatocytes, increased content of total water in the organ, and changed magnetic relaxation properties (spin-lattice and spin-spin relaxation times). Preliminary parasympathetic denervation of the liver (vagotomy) changes the pattern of the organ response to bacterial endotoxin poisoning as indicated by the kinetics of studied morphological and biophysical parameters.