Survival Rate and Enzyme Activities ofLactobacillus acidophilusFollowing Frozen Storage

The ability of two strains of Lactobacillus acidophilus, CRL 640 and CRL 800, to survive and retain their biological activities under frozen storage was determined. Freezing and thawing, as well as frozen storage, damaged the cell membrane, rendering the microorganisms sensitive to sodium chloride and bile salts. Both lactic acid production and proteolytic activity were depressed after 21 days at -20 degreesC, whereas beta-galactosidase activity per cell unit was increased. Cell injury was partially overcome after repair in a salt-rich medium. Copyright 1998 Academic Press.     

Differential modulation of human melanoma cell metalloproteinase expression by alpha2beta1 integrin and CD44 triple-helical ligands derived from type IV collagen

Tumor cell binding to components of the basement membrane is well known to trigger intracellular signaling pathways. Signaling ultimately results in the modulation of gene expression, facilitating metastasis. Type IV collagen is the major structural component of the basement membrane and is known to be a polyvalent ligand, possessing sequences bound by the alpha1beta1, alpha2beta1, and alpha3beta1 integrins, as well as cell surface proteoglycan receptors, such as CD44/chondroitin sulfate proteoglycan (CSPG). The role of alpha2beta1 integrin and CD44/CSPG receptor binding on human melanoma cell activation has been evaluated herein using triple-helical peptide ligands incorporating the alpha1(IV)382-393 and alpha1(IV)1263-1277 sequences, respectively. Gene expression and protein production of matrix metalloproteinases-1 (MMP-1), -2, -3, -13, and -14 were modulated with the alpha2beta1-specific sequence, whereas the CD44-specific sequence yielded significant stimulation of MMP-8 and lower levels of modulation of MMP-1, -2, -13, and -14. Analysis of enzyme activity confirmed different melanoma cell proteolytic potentials based on engagement of either the alpha2beta1 integrin or CD44/CSPG. These results are indicative of specific activation events that tumor cells undergo upon binding to select regions of basement membrane collagen. Based on the present study, triple-helical peptide ligands provide a general approach for monitoring the regulation of proteolysis in cellular systems.     

Production and Analysis of Perdeuterated Lipids from Pichia pastoris Cells

Probing molecules using perdeuteration (i.e deuteration in which all hydrogen atoms are replaced by deuterium) is extremely useful in a wide range of biophysical techniques. In the case of lipids, the synthesis of the biologically relevant unsaturated perdeuterated lipids is challenging and not usually pursued. In this work, perdeuterated phospholipids and sterols from the yeast Pichia pastoris grown in deuterated medium are extracted and analyzed as derivatives by gas chromatography and mass spectrometry respectively. When yeast cells are grown in a deuterated environment, the phospholipid homeostasis is maintained but the fatty acid unsaturation level is modified while the ergosterol synthesis is not affected by the deuterated culture medium. Our results confirm that the production of well defined natural unsaturated perdeuterated lipids is possible and gives also new insights about the process of desaturase enzymes.     

New insights into <Emphasis Type="Italic">SRY</Emphasis> regulation through identification of 5' conserved sequences

Background  

SRY is the pivotal gene initiating male sex determination in most mammals, but how its expression is regulated is still not understood. In this study we derived novel SRY 5' flanking genomic sequence data from bovine and caprine genomic BAC clones.     

MPrime: efficient large scale multiple primer and oligonucleotide design for customized gene microarrays

Background  

Enhancements in sequencing technology have recently yielded assemblies of large genomes including rat, mouse, human, fruit fly, and zebrafish. The availability of large-scale genomic and genic sequence data coupled with advances in microarray technology have made it possible to study the expression of large numbers of sequence products under several different conditions in days where traditional molecular biology techniques might have taken months, or even years. Therefore, to efficiently study a number of gene products associated with a disease, pathway, or other biological process, it is necessary to be able to design primer pairs or oligonucleotides en masse rather than using a time consuming and laborious gene-by-gene method.     

Prion Strain Discrimination Based on Rapid In Vivo Amplification and Analysis by the Cell Panel Assay

Prion strain identification has been hitherto achieved using time-consuming incubation time determinations in one or more mouse lines and elaborate neuropathological assessment. In the present work, we make a detailed study of the properties of PrP-overproducing Tga20 mice. We show that in these mice the four prion strains examined are rapidly and faithfully amplified and can subsequently be discriminated by a cell-based procedure, the Cell Panel Assay.     

Strain-Specific Role of RNAs in Prion Replication

Several lines of evidence suggest that various cofactors may be required for prion replication. PrP binds to polyanions, and RNAs were shown to promote the conversion of PrP(C) into PrP(Sc) in vitro. In the present study, we investigated strain-specific differences in RNA requirement during in vitro conversion and the potential role of RNA as a strain-specifying component of infectious prions. We found that RNase treatment impairs PrP(Sc)-converting activity of 9 murine prion strains by protein misfolding cyclic amplification (PMCA) in a strain-specific fashion. While the addition of RNA restored PMCA conversion efficiency, the effect of synthetic polynucleotides or DNA was strain dependent, showing a different promiscuity of prion strains in cofactor utilization. The biological properties of RML propagated by PMCA under RNA-depleted conditions were compared to those of brain-derived and PMCA material generated in the presence of RNA. Inoculation of RNA-depleted RML in Tga20 mice resulted in an increased incidence of a distinctive disease phenotype characterized by forelimb paresis. However, this abnormal phenotype was not conserved in wild-type mice or upon secondary transmission. Immunohistochemical and cell panel assay analyses of mouse brains did not reveal significant differences between mice injected with the different RML inocula. We conclude that replication under RNA-depleted conditions did not modify RML prion strain properties. Our study cannot, however, exclude small variations of RML properties that would explain the abnormal clinical phenotype observed. We hypothesize that RNA molecules may act as catalysts of prion replication and that variable capacities of distinct prion strains to utilize different cofactors may explain strain-specific dependency upon RNA.     

The role of cholesterol in the activity of pneumolysin, a bacterial protein toxin

The mechanism via which pneumolysin (PLY), a toxin and major virulence factor of the bacterium Streptococcus pneumoniae, binds to its putative receptor, cholesterol, is still poorly understood. We present results from a series of biophysical studies that shed light on the interaction of PLY with cholesterol in solution and in lipid bilayers. PLY lyses cells whose walls contain cholesterol. Using standard hemolytic assays we have demonstrated that the hemolytic activity of PLY is inhibited by cholesterol, partially by ergosterol but not by lanosterol and that the functional stoichiometry of the cholesterol-PLY complex is 1:1. Tryptophan (Trp) fluorescence data recorded during PLY-cholesterol titration studies confirm this ratio, reveal a significant blue shift in the Trp fluorescence peak with increasing cholesterol concentrations indicative of increasing nonpolarity in the Trp environment, consistent with cholesterol binding by the tryptophans, and provide a measure of the affinity of cholesterol binding: K(d) = 400 +/- 100 nM. Finally, we have performed specular neutron reflectivity studies to observe the effect of PLY upon lipid bilayer structure.