The impact of chemokine receptor CX3CR1 deficiency during respiratory infections with Mycobacterium tuberculosis or Francisella tularensis.

Recruitment of immune cells to infection sites is a critical component of the host response to pathogens. This process is facilitated partly through interactions of chemokines with cognate receptors. Here, we examine the importance of fractalkine (CX3CL1) receptor, CX3CR1, which regulates function and trafficking of macrophages and dendritic cells, in the host's ability to control respiratory infections with Mycobacterium tuberculosis or Francisella tularensis. Following low-dose aerosol challenge with M. tuberculosis, CX3CR1(-/-) mice were no more susceptible to infection than wild-type C57BL/6 mice as measured by organ burden and survival time. Similarly, following inhalation of F. tularensis, CX3CR1(-/-) mice displayed similar organ burdens to wild-type mice. CX3CR1(-/-) mice had increased recruitment of monocytes and neutrophils in the lung; however, this did not result in increased abundance of infected monocytes or neutrophils. We conclude that CX3CR1-deficiency affects immune-cell recruitment; however, loss of CX3CR1 alone does not render the host more susceptible to M. tuberculosis or F. tularensis.

NMR studies of a deoxyribodecanucleotide containing an extrahelical thymidine surrounded by an oligo(dA).oligo(dT) tract.

One- and two-dimensional NMR experiments were carried out on a decamer, d-(CGCTTTTCGC).d(GCGAAAAGCG), and on the same sequence with the addition of an unpaired thymidine, d(CGCTTTTCGC).d(GCGAATAAGCG), which will be referred to as the T-bulge decamer. Evidence from one-dimensional NOE experiments on the exchangeable protons indicates that the unpaired thymidine is extrahelical. This conclusion is also supported by numerous cross-peaks in the two-dimensional NOESY spectrum of the nonexchangeable protons. Assignments for all of the resonances, with the exception of the H5' and H5" resonances, have been made for both oligonucleotide duplexes through the use of 2D NOESY, COSY, and relayed COSY experiments. Temperature dependence of the methyl resonance chemical shifts indicates that the unpaired thymidine shows unusual behavior compared to other thymidines in the duplex. Two-dimensional NOESY experiments carried out from 5 to 35 degrees C indicate the unpaired thymidine remains extrahelical throughout this temperature range. A similar temperature dependence for the methyl chemical shift is found in the corresponding single-strand d(GCGAATAAGCG). The oligo-(dA).oligo(dT) tracts in both the decamer and the T-bulge decamer have structures different from B-form DNA and exhibit NOEs similar to those observed in other oligonucleotides containing A.T tracts. The formation of this unusual A.T tract structure may induce the extrahelical conformation of the unpaired thymidine.

NMR study of G.A and A.A pairing in (dGCGAATAAGCG)2.

One- and two-dimensional NMR, UV absorption experiments, and molecular mechanics calculations were conducted on an oligonucleotide duplex (dGCGAATAAGCG)2 which will be referred to as the T-11-mer. This oligonucleotide forms a duplex that is primarily B-form and contains two adjacent G.A and A.A base pairs and two 3' unpaired guanosines. The adjacent mismatch base pairs have an unusual structure which includes overwinding the helix and stacking with the base from the complementary strand (A4 with A8 and G3 with A7) instead of stacking with the base which is sequential on the strand. The exchangeable and nonexchangeable proton NMR spectra of the duplex have been characterized in H2O and D2O solution at neutral and acidic pH. The duplex is stabilized upon protonation; however, no additional hydrogen bonds are formed. We have observed the amino protons of adenosines A4 and A8 and guanosine G3 as a function of temperature and pH. These amino protons are involved in hydrogen bonds with the purine N3 or N7 acting as acceptors. Through the observation of a variety of NOE signals, the structure of the G.A and A.A mismatch base pairs has been defined.