Applications of immortalized cells in basic and clinical neurology

Immortalized cell lines can serve as model systems for studies of neuronal development and restoration of function in models of neurological disease. Cell lines which result from spontaneous or experimentally-induced tumors have been used for these purposes. More recently, the techniques of genetic engineering have resulted in the production of cell lines with specific desired characteristics. This has been accomplished by insertion of a desired gene into a pre-existing immortal cell or by immortalizing primary cells. The production of immortal cell lines using temperature-sensitive immortalizing genes offers an additional method of controlling gene expression, and thereby controlling cell proliferation and differentiation. In the nervous system, these techniques have produced immortal cell lines with neuronal and glial properties.     

Ligand and pathogen specificity of the Atlantic salmon serum C-type lectin

Background

An Atlantic salmon (Salmo salar) C-type lectin (SSL) binds to mannose and related sugars as well as to the surface of Aeromonas salmonicida. To characterize this lectin as a pathogen recognition receptor in salmon, aspects of its interaction with molecules and with intact pathogens were investigated.

Methods

SSL was isolated using whole-yeast-affinity and mannan-affinity chromatography. The binding of SSL to the two major surface molecules of A. salmonicida, lipopolysaccharide (LPS) and A-layer protein was investigated by western blotting and enzyme-linked immunosorbent assays. Microbial binding specificity of SSL was examined by whole cell binding assays using a range of species. Carbohydrate ligand specificity of SSL was examined using glycan array analysis and frontal affinity chromatography.

Results

SSL showed binding to bacteria and yeast including, Pseudomonas fluorescens, A. salmonicida, A. hydrophila, Pichia pastoris, and Saccharomyces cerevisiae, but there was no detectable binding to Yersinia ruckeri. In antimicrobial assays, SSL showed no activity against Escherichia coli, Bacillus subtilis, S. cerevisiae, or A. salmonicida, but it was found to agglutinate E. coli. The major surface molecule of A. salmonicida recognized by SSL was shown to be LPS and not the A-layer protein. LPS binding was mannose-inhibitable. Glycans containing N-acetylglucosamine were shown to be predominant ligands.

Conclusion

SSL has a distinct ligand preference while allowing recognition of a wide variety of related carbohydrate structures.

General Significance

SSL is likely to function as a wide-spectrum pattern recognition protein.     

Production of N 2 O by Ammonia Oxidizing Bacteria at Subfreezing Temperatures as a Model for Assessing the N 2 O Anomalies in the Vostok Ice Core

It was suggested that the abnormally high N ₂ O values found in 130,000–160,000 year-old Vostok ice core samples, characterized by high δ ¹⁵ N and low δ ¹⁸ O values, resulted from in situ microbial N ₂ O production. To substantiate these observations we obtained new geochemical data from the last glacial period and showed the existence of additional small N ₂ O anomalies. To test the hypothesis that microbial metabolism could contribute to these anomalies, we developed protocols for examining the ability of Nitrosomonas cryotolerans cells to produce N ₂ O at subfreezing temperatures. Our results show that these model, frozen cultures produce N ₂ O at temperatures as low as ?32°C.     

Phylogenetic analysis of anaerobic psychrophilic enrichment cultures obtained from a greenland glacier ice core

The examination of microorganisms in glacial ice cores allows the phylogenetic relationships of organisms frozen for thousands of years to be compared with those of current isolates. We developed a method for aseptically sampling a sediment-containing portion of a Greenland ice core that had remained at -9 degrees C for over 100,000 years. Epifluorescence microscopy and flow cytometry results showed that the ice sample contained over 6 x 10(7) cells/ml. Anaerobic enrichment cultures inoculated with melted ice were grown and maintained at -2 degrees C. Genomic DNA extracted from these enrichments was used for the PCR amplification of 16S rRNA genes with bacterial and archaeal primers and the preparation of clone libraries. Approximately 60 bacterial inserts were screened by restriction endonuclease analysis and grouped into 27 unique restriction fragment length polymorphism types, and 24 representative sequences were compared phylogenetically. Diverse sequences representing major phylogenetic groups including alpha, beta, and gamma Proteobacteria as well as relatives of the Thermus, Bacteroides, Eubacterium, and Clostridium groups were found. Sixteen clone sequences were closely related to those from known organisms, with four possibly representing new species. Seven sequences may reflect new genera and were most closely related to sequences obtained only by PCR amplification. One sequence was over 12% distant from its closest relative and may represent a novel order or family. These results show that phylogenetically diverse microorganisms have remained viable within the Greenland ice core for at least 100,000 years.     

Human galectin‐3 interacts with two anticancer drugs

Human galectin‐3 (hGal‐3) is a mammalian lectin involved in regulation of RNA splicing, apoptosis, cell differentiation, and proliferation. Multimerized extracellular hGal‐3 is thought to crosslink cells by binding to glycoproteins and glycosylated cancer antigens on the cell surface or extracellular matrix. Fluorescence spectroscopy and circular dichroism were used to study the interaction of hGal‐3 with two anticancer agents: bohemine and Zn porphyrin (ZnTPPS₄). The dissociation constant (k_D) for binding of bohemine with hGal‐3 was k_D 0.23±0.05 μM. The hyperbolic titration curve indicated the presence of a single bohemine binding site. The binding of ZnTPPS₄ to hGal‐3 (with and without lactose) is of high affinity having k_D=0.18–0.20 μM and is not inhibited by lactose, indicating that ZnTPPS₄ and carbohydrate bind different sites. Circular dichroism spectra of the hGal‐3 complexes suggested that the binding of the hydrophobic compounds changed the hGal‐3 secondary structure. In summary, we show that two compounds with anticancer activity, bohemine and ZnTPPS₄, have high affinity for hGal‐3 at a site that is distinct from its carbohydrate site. Since hGal‐3 binds to several carbohydrate cancer antigens, the results suggest that it may have utility in the targeted delivery of drugs for cancer.     

PA-I lectin from Pseudomonas aeruginosa binds acyl homoserine lactones

The study analyses the binding affinities of Pseudomonas aeruginosa PA-I lectin (PA-IL) to three N-acyl homoserine lactones (AHSL), quorum sensing signal molecules responsible for cell-cell communication in bacteria. It shows that like some plant lectins, PA-IL has a dual function and, besides its carbohydrate-binding capacity, can accommodate AHLS. Formation of complexes between PA-IL and AHSL with acyl side chains composed of 4, 6 or 12 methyl groups is characterized by changes in the emissions of two incorporated fluorescent markers, TNS and IAEDANS, both derivatives of naphthalene sulfonic acid. PA-IL shows increasing affinities to lactones with longer aliphatic side chains. The values of the apparent dissociation constants (K(d)), which are similar to the previously determined K(d) for the adenine high affinity binding, and the similar effects of lactones and adenine on the TNS emission indicate one identical binding site for these ligands, which is suggested to represent the central cavity of the oligomeric molecule formed after the association of the four identical subunits of PA-IL. Intramolecular distances between the fluorescent markers and protein Trp residues are determined by fluorescence resonance energy transfer (FRET).     

Biochemical and immunochemical characterization of boar sperm flagellar protein with role in hyperactivation/capacitation process

Investigations on specific and functionally active sperm antigens could bring about the elucidation of the mechanisms of gamete interaction and help the search for new approaches in prognosis and regulation of fertility. Previously, we reported that the monoclonal antibody (Mab) 3G4 against capacitated boar spermatozoa was capable of inhibiting boar sperm-porcine zona pellucida binding due to its inhibitory effect on sperm hyperactivation and capacitation. The cell and tissue specificity of Mab 3G4 was demonstrated in indirect immunofluorescence (IIF) and ELISA experiments against spermatozoa from different vertebrate species, as well as against extracts of boar reproductive and somatic organs. In the present IIF experiments, it was shown that Mab 3G4 recognized an antigen determinant on the flagellar midpiece region of ejaculated and capacitated boar spermatozoa. It was speculated that the Mab 3G4-corresponding antigen participates in pyruvate/lactate metabolism because of its specific localization in the sperm structure, which is responsible for producing forward motility and its involvement in processes that require the metabolism of pyruvate and lactate. As a possible approach toward investigating the participation of Ag 3G4 in pyruvate/lactate metabolism, Mab 3G4's effect on lactate dehydrogenase (LDH) was examined. Using an electrophoretic approach we provided evidence that Mab 3G4 stimulates LDH activity in the Triton X-100 and NP40 protein fractions of capacitated boar spermatozoa. In addition, we found that LDH isoenzymes stimulated by Mab 3G4 are of gametic C type. In Western blot, under nonreducing conditions, Mab 3G4 identified a single protein band with a molecular weight of 140 kDa. The biochemical and immunochemical experiments provided evidence supporting the involvement of 3G4 antigen in the sperm pyruvate/lactate metabolism.     

Seasonal freeze resistance of rainbow smelt (Osmerus mordax) is generated by differential expression of glycerol-3-phosphate dehydrogenase, phosphoenolpyruvate carboxykinase, and antifreeze protein genes

In winter, rainbow smelt (Osmerus mordax) accumulate glycerol and produce an antifreeze protein (AFP), which both contribute to freeze resistance. The role of differential gene expression in the seasonal pattern of these adaptations was investigated. First, cDNAs encoding smelt and Atlantic salmon (Salmo salar) phosphoenolpyruvate carboxykinase (PEPCK) and smelt glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were cloned so that all sequences required for expression analysis would be available. Using quantitative PCR, expression of beta actin in rainbow smelt liver was compared with that of GAPDH in order to determine its validity as a reference gene. Then, levels of glycerol-3-phosphate dehydrogenase (GPDH), PEPCK, and AFP relative to beta actin were measured in smelt liver over a fall-winter-spring interval. Levels of GPDH mRNA increased in the fall just before plasma glycerol accumulation, implying a driving role in glycerol synthesis. GPDH mRNA levels then declined during winter, well in advance of serum glycerol, suggesting the possibility of GPDH enzyme or glycerol conservation in smelt during the winter months. PEPCK mRNA levels rose in parallel with serum glycerol in the fall, consistent with an increasing requirement for amino acids as metabolic precursors, remained elevated for much of the winter, and then declined in advance of the decline in plasma glycerol. AFP mRNA was elevated at the onset of fall sampling in October and remained elevated until April, implying separate regulation from GPDH and PEPCK. Thus, winter freezing point depression in smelt appears to result from a seasonal cycle of GPDH gene expression, with an ensuing increase in the expression of PEPCK, and a similar but independent cycle of AFP gene expression.