Effects of establishing cell cultures and cell culture conditions on the proliferative life span of human fibroblasts isolated from different tissues and donors of different ages

It is well known that normal human cells placed in a culture environment exhibit a limited proliferative capacity. The extent to which the culture environment influences proliferative life span is not understood. This study evaluated the effects of the standard procedures used to establish and maintain cultures on the proliferative life spans of different types of human fibroblast cells established from fetal and adult skin and lung. The results of this study demonstrate that procedures to establish cell cultures use only one of several subpopulations of cells present in biopsy pieces and that the culture conditions routinely employed by most laboratories can exert significant effects on proliferative life-span determinations. The maximum proliferative life span differed significantly when obtained by growing the cells in two commonly used commercial media. Proliferative life span was inversely related to ambient oxygen tension and directly related to seeding density in all of the lines examined although lines established from adult skin were much more resistant to toxicity. Enzymatic antioxidant defense levels of fetal skin fibroblasts were much lower than those observed in adult skin fibroblasts, but the effects of oxygen on their life spans were similar. Hyperoxia induced larger increases in glutathione concentration in cell lines with low antioxidant enzyme levels.     

Point mutants of EHEC intimin that diminish Tir recognition and actin pedestal formation highlight a putative Tir binding pocket

Attachment to host cells by enterohaemorrhagic Escherichia coli (EHEC) is associated with the formation of a highly organized cytoskeletal structure containing filamentous actin, termed an attaching and effacing (AE) lesion. Intimin, an outer membrane protein of EHEC, is required for the formation of AE lesions, as is Tir, a bacterial protein that is translocated into the host cell to function as a receptor for intimin. We established a yeast two-hybrid assay for intimin-Tir interaction and, after random mutagenesis, isolated 24 point mutants in intimin, which disrupted Tir recognition in this system. Analysis of 11 point mutants revealed a correlation between recognition of recombinant Tir and the ability to trigger AE lesions. Many of the mutations fell within a 50-residue region near the C-terminus of intimin. Alanine-scanning mutagenesis of this region revealed four residues (Ser890, Thr909, Asn916 and Asn927) that are critical for Tir recognition. Mapping the sequences of EHEC intimin and Tir onto the crystal structure of the intimin-Tir complex of enteropathogenic E. coli predicts that each of these four intimin residues lies at the intimin-Tir interface and contributes to a pocket that interacts with Ile298 of EHEC Tir. Thus, this genetic approach to intimin function both identified residues critical for Tir binding and demonstrated a correlation between the ability to bind Tir and the ability to trigger actin focusing.     

Genetic and physical mapping of a rice blast resistance locus,Pi-CO39(t), that corresponds to the avirulence gene AVR1-CO39 of Magnaporthe grisea

We have identified, genetically mapped and physically delineated the chromosomal location of a new rice blast resistance locus, designated Pi-CO39(t). This locus confers resistance to Magnaporthe grisea isolates carrying the AVR1-CO39 avirulence locus. The AVR1-CO39 locus is conserved in non-rice (cereals and grasses)-infecting isolates of M. grisea, making Pi-CO39(t) useful for engineering M. grisea resistance in rice and other cereals. The resistance in the rice line CO39 was inherited as a single dominant locus in segregating populations derived from F(2) and F(3) crosses between disease-resistant (CO39) and susceptible (51583) rice genotypes. Microsatellite, RFLP and resistance gene analog (RGA) markers were used to map the Pi-CO39(t) locus to a 1.2-cM interval between the probenazole-responsive ( RPR1) gene (0.2 cM) and RFLP marker S2712 (1.0 cM) on the short arm of rice chromosome 11. RFLP markers G320 and F5003, and resistance gene analogs RGA8, RGA38 and RGACO39 were tightly linked to the Pi-CO39(t) locus (no recombination detected in a sample of ~2400 gametes). A large-insert genomic library of CO39 was constructed in the binary plant transformation vector pCLD04541. A library screen using RGA8, RGA38 and probes derived from the ends of CO39 clones, as well as BAC end probes from the corresponding locus in the rice cv. Nipponbare, resulted in the assembly of three CO39 contigs of 180 kb, 110 kb and 145 kb linked to the Pi-CO39(t) locus. A 650-kb contig was also constructed representing the susceptible locus, pi-CO39(t), in the Nipponbare genome. The two genomes are highly divergent with respect to additions, deletions and translocations at the Pi-CO39(t) locus, as revealed by the presence or absence of mapping markers.     

Obtaining the shear stress versus shear rate relationship and yield stress of blood from capillary viscometry data by Tikhonov regularization

This paper describes a procedure, based on Tikhonov regularization, for extracting the shear stress versus shear rate relationship and yield stress of blood from capillary viscometry data. The relevant equations and the mathematical nature of the problem are briefly described. The procedure is then applied to three sets of capillary viscometry data of blood taken from the literature. From each data set the procedure computes the complete shear stress versus shear rate relationship and the yield stress. Since the procedure does not rely on any assumed constitutive equation, the computed rheological properties are therefore model-independent. These properties are compared against one another and against independent measurements. They are found to be in good agreement for shear stress greater than 0.1 Pa but show significant deviations for shear stress below this level. A possible way of improving this situation is discussed.     

Study of trace element correlations with drought tolerance in different sorghum genotypes using energy-dispersive X-ray fluorescence technique

Drought-tolerant and drought-susceptible genotypes of sorghum (Sorghum bicolor L. Monech) were analyzed by the energy-dispersive X-ray fluorescence (EDXRF) technique to study the correlation of trace elements with drought-tolerance capacities. Samples prepared from mature seeds, young seedlings, and old plants were analyzed using a ¹⁰⁹Cd radioisotope source and a Si(Li) semiconductor detector of resolution 170 eV for 5.9-keV MnK{ie255-1} X-rays. Elements such as K, Fe, Cu, Zn, Rb and Sr and Y were found to be present in varying concentrations in different samples. The trace element profile studied in the seeds of 11 genotypes and in seedlings (young and old) of 4 sorghum genotypes showed considerable variation. The genotype Arfa Gadamak (AG) showed a distinct presence of a high level of Zn in its young seedling. It was observed that in most of the genotypes (seeds), K and Fe concentrations were more in the tolerant genotype as compared to the susceptible type. The concentration of Fe decreased with maturity in the tolerant group and it increased with maturity in the susceptible group.     

Tails of two Tirs: actin pedestal formation by enteropathogenic E. coli and enterohemorrhagic E. coli O157:H7

Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli O157:H7 (EHEC) form characteristic lesions on infected mammalian cells called actin pedestals. Each of these two pathogens injects its own translocated intimin receptor (Tir) molecule into the plasma membranes of host cells. Interaction of translocated Tir with the bacterial outer membrane protein intimin is required to trigger the assembly of actin into focused pedestals beneath bound bacteria. Despite similarities between the Tir molecules and the host components that associate with pedestals, recent work indicates that EPEC and EHEC Tir are not functionally interchangeable. For EPEC, Tir-mediated binding of Nck, a host adaptor protein implicated in actin signaling, is both necessary and sufficient to initiate actin assembly. In contrast, for EHEC, pedestals are formed independently of Nck, and require translocation of bacterial factors in addition to Tir to trigger actin signaling.     

Characterization of two phosphate transporters from barley; evidence for diverse function and kinetic properties among members of the Pht1 family

Putative phosphate transporters have been identified in a barley (Hordeum vulgare L.) genomic library by their homology to known phosphate transporters from dicot species. The genes designated HORvu;Pht1;1 and HORvu;Pht1;6 encode proteins of 521 and 535 amino acids respectively with 12 predicted membrane-spanning domains and other motifs common to the Phtl family of phosphate transporters. HORvu;Pht1;1 is expressed exclusively in roots and is strongly induced by phosphate deprivation. HORvu;Pht1;6 is expressed in the aerial parts of the plant with strongest expression in old leaves and flag leaves. In situ hybridization showed that HORvu;Pht1;6 is expressed in the phloem of vascular bundles in leaves and ears. In order to study the biochemical properties of HORvu;Pht1;1 and HORvu;Pht1;6, the genes were expressed in transgenic rice (Oryza sativa L.) plants under the control of the rice actin promoter and suspension cell cultures were generated. Cells derived from transgenic plants were able to take up phosphate at a much higher rate than control cells, demonstrating that both genes encode functional phosphate transporters. The estimated Km for phosphate for cells expressing HORvu;Pht1;1 was 9.06 +/- 0.82 microM, which is characteristic of a high-affinity transporter. The rate of phosphate uptake decreased with increasing pH, suggesting that HORvu;Pht1;1 operates as a H+/H2PO4(-) symporter. In contrast, the estimated Km for phosphate for cells expressing HORvu;Pht1;6 was 385 +/- 61 microM, which is characteristic of a low-affinity transporter. Taken together, the results suggest that HORvu;Pht1;1 functions in uptake of phosphate at the root surface, while HORvu;Pht1;6 probably functions in remobilization of stored phosphate from leaves.     

Liquid chromatography-tandem mass spectrometric quantitative determination of the HIV protease inhibitor atazanavir (BMS-232632) in human peripheral blood mononuclear cells (PBMC): practical approaches to PBMC preparation and PBMC assay design for high-throughput analysis

A selective, accurate, and reproducible LC/MS/MS assay was developed and validated for the determination of the HIV protease inhibitor atazanavir (BMS-232632) in human peripheral blood mononuclear cells (PBMC) samples. In addition to the details of the validated LC/MS/MS method, a practical procedure is described in great detail for the preparation of large supplies of control (blank) PBMC from units of blood (each unit of blood is about 500 ml) for making the calibration standards and quality control (QC) samples. The PBMC assay design, intended for high-throughput sample analysis, is also described in some detail in regards to the composition and concentration expressions of the calibration standards and QC samples, the lysing procedure of the PBMC samples, and the final analysis/quantitation procedure. The method involved automated solid-phase extraction (SPE) of atazanavir and a stable isotope analog internal standard (I.S.) using 3M Empore C2-SD 96-well plates. A portion of the reconstituted sample residue was injected onto a YMC Basic analytical column which was connected to a triple quad mass spectrometer for analyte determination by positive-ion electrospray in the selected reaction monitoring (SRM) mode. The standard curve, which ranged from 5 to 2500 fmol per one million cells (fmol/10(6) cells), was fitted to a quadratic regression model weighted by 1/concentration. The lower limit of quantitation (LLOQ) was 5 fmol/10(6) cells. The inter- and intra-run coefficients of variation (CV) for the assay were <9% and the accuracy was 94-104%. Atazanavir was stable in PBMC for at least 24h at room temperature and for at least 129 days at -15 degrees C.