New differentiation markers of mouse liver epithelial cell lines

We describe two new markers of mouse liver epithelial cells detected by monoclonal antibodies. Immunomorphological localization of antigens was performed using light and electron microscopy. Antigen G7 is a marker of cholangiocytes and oval cells. Antigen A6 is present in cholangiocytes and oval cells; moreover, it is expressed in normal liver in single hepatocytes adjacent to the portal vein, in preneoplastic liver, in newly formed hepatocytes, and in certain hepatocarcinomas. Thus, antigen A6 is a marker of cholangiocytes, oval cells and of certain stages of hepatocyte differentiation. We also detected phenotypic heterogeneity of Gehring cells in terms of antigen A6 content. We have formulated problem of the relationship between A6-negative Gehring cells and liver stem cells. Both marker antigens are species-specific but are not specific for the liver. Antigen A6 is simultaneously a differentiation marker of cells belonging to the erythroid series. It is expressed in erythroblasts of fetal liver and is absent in erythroblasts of the yolk sac and erythrocytes. The relationship between antigen A6 and blood group antigens is discussed.     

Cloning and sequencing of immunoglobin lambda-chain cDNA in american mink (Mustela vison

cDNA library in the lambda gt11 phage was constructed using poly (A+)-mRNA from mink spleen as a template. Immunoscreening of the library allowed the identification of 2 lambda-related clones containing 370 and 803 bp insertion (lambda IGL-1 and lambda IGL-2). Analysis of the primary structure of lambda IGL-2 demonstrated that it contains a large portion of V lambda-segment, J lambda-segment, C lambda-gene and its 3'-untranslated part. The nucleotide sequences known for the immunoglobulin genes were compared to the sequence of the lambda IGL-2 clone. The highest degree of homology was established for the rabbit lambda-genes, this being 63, 94 and 72% for the RF3 region of V lambda-segment, J lambda-segment and C lambda-region, respectively.     

Mink IgG-allotypes and Aleutian disease

IgG polymorphism (allotypes H3, H4, H6 and H8 of constant region of the gamma-chain) was investigated in healthy and affected with Aleutian disease (AD) minks from two Siberian and one Danish populations. In all three populations, the expression of H3 and H4 allotypes was strongly associated with AD. Among the AD minks the frequency of H6, H8 phenotype was found to be decreased, whereas the frequency of H3, H4, H6, H8 phenotype was significantly increased. At the same time, the populational distribution of the rest phenotypes was similar among healthy and AD minks. The H3, H4, H6, H8 minks showed the highest pathomorphological characteristics of AD. Based on the data concerning mink H3 and H4, and human Gm allotypes, their role as possible genetic markers for hereditary susceptibility to distinct disease is discussed.     

Immunogenetic study on the polymorphism of serum α2-lipoprotein in mink. III. Ld1 allotype of low-density lipoprotein

Mink Ld1 antigen of serum low-density lipoprotein was demonstrated by alloantibodies. No genetic relation was found between Ld1 and the Lpm system of very-high-density lipoprotein. The existence of an autosomal dominant gene, coding for the new alloantigenic marker, is postulated on the basis of mink breeding data.     

Identification and genetic control of the Ld1-allotype of mink low-density serum lipoprotein]

Ld1, an antigen of serum low density lipoprotein, was identified by means of mink alloimmunization. No genetic relation was established between Ld1 and the Ipm-system of very high density lipoprotein. Based on the analysis of breeding data, the existence of an autosomal dominant gene, Ld1+, is postulated. This gene codes for the new alloantigenic marker. The existence of at least one other allotype of the Ld-system is suggested.     

3 new allotypes of mink blood serum alpha2-lipoproteins--Lpm 6, Lpm 7, Lpm 8]

Three new allotypes were discovered in mink sera by means of isoimmunization. Based on the results of identifications, they were referred to the Lpm system of serum alpha2-lipoprotein. They were designated as Lpm 6, Lpm 7 and Lpm 8. The allotyping of serum samples from 342 minks made possible to establish a relation between Lpm 7 and Lpm 8 using the chi2 method; it was also found that these two allotypes related to each of the first five Lpm as well as to their phenotypes, which was described earlier. There was a significant dependence of the expression of Lpm 6 on Lpm 5 indicating a genetic relation between Lpm 6 and other allotypes. The detection of Lpm 6, 7 and 8, which are the most likely under the control of the same gene (or a sustem of genes) as Lpm 1, 2, 3 and 5, is an evidence for the complex structure of the Lpm locus.     

Halotolerant, alkaliphilic urease-producing bacteria from different climate zones and their application for biocementation of sand

Microbially induced calcium carbonate precipitation (MICP) is a phenomenon based on urease activity of halotolerant and alkaliphilic microorganisms that can be used for the soil bioclogging and biocementation in geotechnical engineering. However, enrichment cultures produced from indigenous soil bacteria cannot be used for large-scale MICP because their urease activity decreased with the rate about 5 % per one generation. To ensure stability of urease activity in biocement, halotolerant and alkaliphilic strains of urease-producing bacteria for soil biocementation were isolated from either sandy soil or high salinity water in different climate zones. The strain Bacillus sp. VUK5, isolated from soil in Ukraine (continental climate), was phylogenetically close in identity (99 % of 16S rRNA gene sequence) to the strain of Bacillus sp. VS1 isolated from beach sand in Singapore (tropical rainforest climate), as well as to the strains of Bacillus sp. isolated by other researchers in Ghent, Belgium (maritime temperate climate) and Yogyakarta, Indonesia (tropical rainforest climate). Both strains Bacillus sp. VS1 and VUK5 had maximum specific growth rate of 0.09/h and maximum urease activities of 6.2 and 8.8 mM of hydrolysed urea/min, respectively. The halotolerant and alkaliphilic strain of urease-producing bacteria isolated from water of the saline lake Dead Sea in Jordan was presented by Gram-positive cocci close to the species Staphylococcus succinus. However, the strains of this species could be hemolytic and toxigenic, therefore only representatives of alkaliphilic Bacillus sp. were used for the biocementation studies. Unconfined compressive strengths for dry biocemented sand samples after six batch treatments with strains VS1and VUK5 were 765 and 845 kPa, respectively. The content of precipitated calcium and the strength of dry biocemented sand at permeability equals to 1 % of initial value were 12.4 g Ca/kg of dry sand and 454 kPa, respectively, in case of biocementation by the strain VS1. So, halotolerant, alkaliphilic, urease-producing bacteria isolated from different climate zones have similar properties and can be used for biocementation of soil.     

Improving performance of docking-based virtual screening by structural filtration

In the current study an innovative method of structural filtration of docked ligand poses is introduced and applied to improve the virtual screening results. The structural filter is defined by a protein-specific set of interactions that are a) structurally conserved in available structures of a particular protein with its bound ligands, and b) that can be viewed as playing the crucial role in protein-ligand binding. The concept was evaluated on a set of 10 diverse proteins, for which the corresponding structural filters were developed and applied to the results of virtual screening obtained with the Lead Finder software. The application of structural filtration resulted in a considerable improvement of the enrichment factor ranging from several folds to hundreds folds depending on the protein target. It appeared that the structural filtration had effectively repaired the deficiencies of the scoring functions that used to overestimate decoy binding, resulting into a considerably lower false positive rate. In addition, the structural filters were also effective in dealing with some deficiencies of the protein structure models that would lead to false negative predictions otherwise. The ability of structural filtration to recover relatively small but specifically bound molecules creates promises for the application of this technology in the fragment-based drug discovery.     

Glycosylated equine prolactin and its carbohydrate moiety

Glycosylated equine prolactin (G-ePRL) and nonglycosylated ePRL were purified to homogeneity from side fractions obtained during isolation of LH/FSH from horse pituitaries. Both PRL forms were isolated together in high yield by the isolation procedure used for glycosylated porcine PRL/(G-pPRL) and pPRL, involving acetone extraction/precipitation, NaCl and isoelectric precipitation, and gel filtration. Purification of G-ePRL required additional Con A chromatography. The N-terminal amino acid sequencing for 32 cycles of G-ePRL and ePRL resulted in sequences identical to the known primary structure of ePRL. Based on MALDI mass spectrometry analysis and SDS-PAGE mobilities,G-ePRL and ePRL had estimated molecular weights of 25,000 and 23,000 Da, respectively. G-ePRL displayed only 60% of the immunoreactivity of ePRL in homologous radioimmunoassay. Using the Nb2 lymphoma cell bioassay, ePRL was found to have about l/30th the mitogenic activity of bovine PRL; G-ePRL was approximately l/10th as active as ePRL. Glycosylation of G-ePRL at Asn³¹ was confirmed by isolation and sequence analysis of an enzymatically derived G-ePRL glycopeptide spanning residues 29–37. Monosaccharide compositions of intact G-ePRL and this glycopeptide were very similar (Man₃, GlcNAc₂, GalNAc₁, Fuc_0.6, Gal_0.2, NeuAc_0.15) and resembled that of G-pPRL. The glycopeptide contained one sulfate residue as determined by ion chromatography after acid hydrolysis, indicating the presence of a sulfated monosaccharide. Comparative carbohydrate analysis of G-ePRL and other G-PRL preparations suggests that the functionally significant Asn³¹ carbohydrate unit is a fucosylated complex mono- and/or biantennary oligosaccharide terminating with a sulfated GalNAc residue and two or three Man residues.