The primary structure of the beta-subunit of the cell surface adhesion glycoproteins LFA-1, CR3 and p150,95 and its relationship to the fibronectin receptor.

The lymphocyte-function-associated antigen-1 (LFA-1), the complement receptor type 3 (CR3) and the antigen p150,95 are cell-surface glycoproteins. They are heterodimeric complexes, each containing a unique alpha-subunit noncovalently associated with a common beta-subunit. We have purified the beta-subunit from human spleen and obtained limited peptide sequences. What appears to be the complete primary structure for the fully processed beta-subunit was obtained by cDNA sequencing of clones from a phorbol ester (PMA) stimulated U937 cDNA library. There are five possible glycosylation sites and a transmembrane segment. The sequence contains a high level of cysteine (7.6%), with 24 of the 57 cysteine residues being found in three repeating units each with eight residues. The entire primary structure has 47% identity to a subunit of a fibronectin binding protein from chicken fibroblasts. It seems that LFA-1, CR3 and p150,95 antigens may belong to an extended family of cell surface molecules including the fibronectin binding protein.     

Distribution and Expression of Elicitin Genes in the Interspecific Hybrid Oomycete Phytophthora alni

Phytophthora alni subsp. alni, P. alni subsp. multiformis, and P. alni subsp. uniformis are responsible for alder disease in Europe. Class I and II elicitin gene patterns of P. alni subsp. alni, P. alni subsp. multiformis, P. alni subsp. uniformis, and the phylogenetically close species P. cambivora and P. fragariae were studied through mRNA sequencing and 3′ untranslated region (3′UTR)-specific PCRs and sequencing. The occurrence of multiple 3′UTR sequences in association with identical elicitin-encoding sequences in P. alni subsp. alni indicated duplication/recombination events. The mRNA pattern displayed by P. alni subsp. alni demonstrated that elicitin genes from all the parental genomes are actually expressed in this allopolyploid taxon. The complementary elicitin patterns resolved confirmed the possible involvement of P. alni subsp. multiformis and P. alni subsp. uniformis in the genesis of the hybrid species P. alni subsp. alni. The occurrence of multiple and common elicitin gene sequences throughout P. cambivora, P. fragariae, and P. alni sensu lato, not observed in other Phytophthora species, suggests that duplication of these genes occurred before the radiation of these species.     

Sex differences in circulating antibodies in nestling Pied Flycatchers Ficedula hypoleuca

Sex differences in immune function are relatively well studied in vertebrate animals, although the patterns are not always clear in birds. The study of immune responses in nestlings of wild bird populations may constitute an appropriate way to investigate inherent intersexual differences while controlling for environmental conditions such as parasitism that affect male and female individuals growing in the same nest. We studied whether the cell‐mediated immune response, as measured by phytohaemagglutinin (PHA) injection, and the levels of circulating antibodies differ between sexes of Pied Flycatcher nestlings Ficedula hypoleuca. No sex differences in nestling cell‐mediated immune response were found, but females showed significantly higher levels of plasma immunoglobulins than males did. Although nestling birds may not have a fully functional humoral immune defence, our study indicates that sex differences in the humoral component exist at this early stage of life. Given the importance of antibodies in the fight against parasite, bacterial and viral infections, the intrinsic sex disparity in circulating antibodies may have important implications for the life history of each sex.     

Possible role of the membrane Na+/H+ antiport in ornithine decarboxylase induction by L-asparagine

Amino acids of transport systems A and N play certain important role in cell activation. For example, the presence of these amino acids is essential in the induction of ornithine decarboxylase by growth factors and hormones. At mM concentrations, each of these amino acids, particularly L-asparagine, can also induce the enzyme without being further metabolized or incorporated into proteins. We have reported that the addition of 10 mM L-asparagine to quiescent Reuber's H-35 rat hepatoma cells caused an immediate and transient increase in intracellular pH. Here we report that concomitant with the intracellular alkalinization was an increase in H+ extrusion which was amiloride-sensitive and Na+-dependent. The induction of ornithine decarboxylase by L-asparagine was also amiloride-sensitive.     

Effects of Polyamines on the Uptake of Neurotransmitters by Rat Brain Synaptosomes

Abstract: The influence of putrescine, spermidine, spermine, and some aliphatic α,ω-diamines on the uptake of neurotransmitters by rat forebrain synaptosomes was investigated. Choline uptake was most effectively inhibited by spermine (IC₅₀= 0.22 m M ), less so by spermidine (IC₅₀= 4.0 m M ), but not by putrescine (IC₅₀ > 100 m M ). At 10 m M, 1,3-diaminopropane, cadaverine, and 1,8-diaminooctane all inhibited choline uptake by 50% or more. Spermine and spermidine inhibited the uptake of dopamine with IC₅₀ values of 2.7 and 2.2 m M , respectively. Putrescine was only slightly inhibitory (IC₅₀= 17.3 m M ) and the other diamines were inactive. The uptake of γ-aminobutyrate (GABA) was only slightly inhibited (15–40%) by the polyamines at 10 m M . With the exception of inhibition of glycine uptake by 1,8-diaminooctane (60%) and of glutamate uptake by cadaverine (35%) none of the polyamines, tested at 10 m M , affected the uptake of adenosine, glutamate, and glycine significantly. A possible modulatory role for polyamines in synaptic transmission through interaction by negatively charged groups of the synaptic membrane with the polycationic compounds is discussed.     

Comparison of Methods for Estimating Bird Abundance and Trends From Historical Count Data

Abstract: The use of bird counts as indices has come under increasing scrutiny because assumptions concerning detection probabilities may not be met, but there also seems to be some resistance to use of model-based approaches to estimating abundance. We used data from the United States Forest Service, Southern Region bird monitoring program to compare several common approaches for estimating annual abundance or indices and population trends from point-count data. We compared indices of abundance estimated as annual means of counts and from a mixed-Poisson model to abundance estimates from a count-removal model with 3 time intervals and a distance model with 3 distance bands. We compared trend estimates calculated from an autoregressive, exponential model fit to annual abundance estimates from the above methods and also by estimating trend directly by treating year as a continuous covariate in the mixed-Poisson model. We produced estimates for 6 forest songbirds based on an average of 621 and 459 points in 2 physiographic areas from 1997 to 2004. There was strong evidence that detection probabilities varied among species and years. Nevertheless, there was good overall agreement across trend estimates from the 5 methods for 9 of 12 comparisons. In 3 of 12 comparisons, however, patterns in detection probabilities potentially confounded interpretation of uncorrected counts. Estimates of detection probabilities differed greatly between removal and distance models, likely because the methods estimated different components of detection probability and the data collection was not optimally designed for either method. Given that detection probabilities often vary among species, years, and observers investigators should address detection probability in their surveys, whether it be by estimation of probability of detection and abundance, estimation of effects of key covariates when modeling count as an index of abundance, or through design-based methods to standardize these effects.     

Lipoteichoic acid is an important microbe-associated molecular pattern of Lactobacillus rhamnosus GG

Background

Receptors with a single transmembrane (TM) domain are essential for the signal transduction across the cell membrane. NMR spectroscopy is a powerful tool to study structure of the single TM domain. The expression and purification of a TM domain in Escherichia coli (E.coli) is challenging due to its small molecular weight. Although ketosteroid isomerase (KSI) is a commonly used affinity tag for expression and purification of short peptides, KSI tag needs to be removed with the toxic reagent cyanogen bromide (CNBr).

Result

The purification of the TM domain of p75 neurotrophin receptor using a KSI tag with the introduction of a thrombin cleavage site is described herein. The recombinant fusion protein was refolded into micelles and was cleaved with thrombin. Studies showed that purified protein could be used for structural study using NMR spectroscopy.

Conclusions

These results provide another strategy for obtaining a single TM domain for structural studies without using toxic chemical digestion or acid to remove the fusion tag. The purified TM domain of p75 neurotrophin receptor will be useful for structural studies.     

NSC666715 and Its Analogs Inhibit Strand-Displacement Activity of DNA Polymerase β and Potentiate Temozolomide-Induced DNA Damage,Senescence and Apoptosis in Colorectal Cancer Cells

Recently approved chemotherapeutic agents to treat colorectal cancer (CRC) have made some impact; however, there is an urgent need for newer targeted agents and strategies to circumvent CRC growth and metastasis. CRC frequently exhibits natural resistance to chemotherapy and those who do respond initially later acquire drug resistance. A mechanism to potentially sensitize CRC cells is by blocking the DNA polymerase β (Pol-β) activity. Temozolomide (TMZ), an alkylating agent, and other DNA-interacting agents exert DNA damage primarily repaired by a Pol-β-directed base excision repair (BER) pathway. In previous studies, we used structure-based molecular docking of Pol-β and identified a potent small molecule inhibitor (NSC666715). In the present study, we have determined the mechanism by which NSC666715 and its analogs block Fen1-induced strand-displacement activity of Pol-β-directed LP-BER, cause apurinic/apyrimidinic (AP) site accumulation and induce S-phase cell cycle arrest. Induction of S-phase cell cycle arrest leads to senescence and apoptosis of CRC cells through the p53/p21 pathway. Our initial findings also show a 10-fold reduction of the IC₅₀ of TMZ when combined with NSC666715. These results provide a guide for the development of a target-defined strategy for CRC chemotherapy that will be based on the mechanisms of action of NSC666715 and TMZ. This combination strategy can be used as a framework to further reduce the TMZ dosages and resistance in CRC patients.