Functional evidence for a small and rigid active site in a high fidelity DNA polymerase: probing T7 DNA polymerase with variably sized base pairs

Hypotheses on the origins of high fidelity in replicative DNA polymerases have recently focused on the importance of geometric or steric effects in this selectivity. Here we reported a systematic study of the effects of base pair size in T7 DNA polymerase (pol), the replicative enzyme for bacteriophage T7. We varied base pair size in very small (0.25 A) increments by use of a series of nonpolar thymidine shape mimics having gradually increasing size. Steady-state kinetics were evaluated for the 5A7A exonuclease-deficient mutant in a 1:1 complex with thioredoxin. For T7 pol, we studied insertion of natural nucleotides opposite variably sized T analogs in the template and, conversely, for variably sized dTTP analogs opposite natural template bases. The enzyme displayed extremely high selectivity for a specific base pair size, with drops in efficiency of as much as 280-fold for increases of 0.4 A beyond an optimum size approximating the size of a natural pair. The enzyme also strongly rejected pairs that were smaller than the optimum by as little as 0.3 A. The size preferences with T7 DNA pol were generally smaller, and the steric rejection was greater than DNA pol I Klenow fragment, correlating with the higher fidelity of the former. The hypothetical effects of varied active site size and rigidity are discussed. The data lend direct support to the concept that active site tightness is a chief determinant of high fidelity of replicative polymerases and that a less rigid (looser) and larger active site can lead to lower fidelity.     

Imaging and automated detection of Sitophilus oryzae (Coleoptera: Curculionidae) pupae in hard red winter wheat

Computed tomography, an imaging technique commonly used for diagnosing internal human health ailments, uses multiple x-rays and sophisticated software to recreate a cross-sectional representation of a subject. The use of this technique to image hard red winter wheat, Triticum aestivm L., samples infested with pupae of Sitophilus oryzae (L.) was investigated. A software program was developed to rapidly recognize and quantify the infested kernels. Samples were imaged in a 7.6-cm (o.d.) plastic tube containing 0, 50, or 100 infested kernels per kg of wheat. Interkernel spaces were filled with corn oil so as to increase the contrast between voids inside kernels and voids among kernels. Automated image processing, using a custom C language software program, was conducted separately on each 100 g portion of the prepared samples. The average detection accuracy in the five infested kernels per 100-g samples was 94.4 +/- 7.3% (mean +/- SD, n = 10), whereas the average detection accuracy in the 10 infested kernels per 100-g sample was 87.3 +/- 7.9% (n = 10). Detection accuracy in the 10 infested kernels per 100-g samples was slightly less than the five infested kernels per 100-g samples because of some infested kernels overlapping with each other or air bubbles in the oil. A mean of 1.2 +/- 0.9 (n = 10) bubbles (per tube) was incorrectly classed as infested kernels in replicates containing no infested kernels. In light of these positive results, future studies should be conducted using additional grains, insect species, and life stages.     

Matrix metalloproteinase-9 deficiency impairs host defense against abdominal sepsis

Matrix metalloproteinase (MMP)-9 is involved in extracellular matrix degradation and leukocyte migration. To determine the role of MMP-9 in the innate immune response to peritonitis, MMP-9 gene-deficient (MMP-9(-/-)) and normal wild-type mice were i.p. infected with Escherichia coli. MMP-9 mRNA and pro-MMP-9 protein levels increased rapidly upon induction of peritonitis. Although MMP-9(-/-) neutrophils showed a normal phagocytosis of E. coli in vitro, MMP-9(-/-) mice displayed a reduced resistance against E. coli peritonitis, as indicated by an enhanced bacterial outgrowth in the peritoneal cavity and increased dissemination of the infection. Furthermore, the cytokine response to LPS was not influenced by MMP-9 deficiency. However, during E. coli peritonitis, MMP-9(-/-) mice showed much higher peritoneal chemokine and cytokine levels compared with wild-type mice. Despite the increased local chemokine concentrations, MMP-9(-/-) mice displayed a diminished recruitment of leukocytes to the site of infection, indicating that cellular migration was impaired. Moreover, MMP-9(-/-) mice developed more severe distant organ damage during infection. These data suggest that MMP-9 is an essential component of an effective host response to E. coli peritonitis.     

Endogenous tissue-type plasminogen activator is protective during Escherichia coli-induced abdominal sepsis in mice

Sepsis is associated with enhanced production of tissue-type plasminogen activator (tPA). We investigated the function of endogenous tPA in the immune responses to Escherichia coli-induced abdominal sepsis using tPA gene-deficient (tPA(-/-)) and normal wild-type (WT) mice. tPA(-/-) mice demonstrated an impaired defense against E. coli peritonitis as indicated by higher bacterial loads at the primary site of the infection, enhanced dissemination, and reduced survival. The protective function of tPA was independent of plasmin since plasminogen gene-deficient (Plg(-/-)) mice were indistinguishable from WT mice. Relative to WT mice, tPA(-/-) mice demonstrated similar neutrophil counts in the peritoneal cavity despite much higher bacterial loads and higher local concentrations of neutrophil attracting chemokines, suggesting a reduced migratory response. In line, tPA(-/-) mice demonstrated a reduced thioglycolate-induced neutrophil influx into the peritoneal cavity and i.p. injection of WT mice with a replication-defective adenoviral vector expressing tPA caused an enhanced cell migration to the peritoneal cavity during E. coli peritonitis. These findings identify a novel protective function of tPA in abdominal sepsis caused by E. coli that seems independent of its role in the generation of plasmin.     

Pulmonary lipopolysaccharide (LPS)-binding protein inhibits the LPS-induced lung inflammation in vivo

LPS-binding protein (LBP) facilitates the interaction of the Gram-negative cell wall component LPS with CD14, thereby enhancing the immune response to LPS. Although lung epithelial cells have been reported to produce LBP in vitro, knowledge of the in vivo role of pulmonary LBP is limited. Therefore, in the present study we sought to determine the function of pulmonary LBP in lung inflammation induced by intranasal administration of LPS in vivo. Using LBP-deficient (LBP-/-) and normal wild-type mice, we show that the contribution of LBP to pulmonary LPS responsiveness depended entirely on the LPS dose. Although the inflammatory response to low dose (1 ng) LPS was attenuated in LBP-/- mice, neutrophil influx and cytokine/chemokine concentrations in the bronchoalveolar compartment were enhanced in LBP-/- mice treated with higher (>10 ng) LPS doses. This finding was specific for LBP, because the exogenous administration of LBP to LBP-/- mice reversed this phenotype and reduced the local inflammatory response to higher LPS doses. Our results indicate that pulmonary LBP acts as an important modulator of the LPS response in the respiratory tract in vivo. This newly identified function of pulmonary LBP might prove beneficial by enabling a protective immune response to low LPS doses while preventing an overwhelming, potentially harmful immune response to higher doses of LPS.     

Monokine induced by interferon gamma and IFN-gamma response to a fusion protein of Mycobacterium tuberculosis ESAT-6 and CFP-10 in Brazilian tuberculosis patients

IFN-gamma responses to Mycobacterium tuberculosis antigens ESAT-6 and CFP-10 have been proposed as specific markers of M. tuberculosis infection. Monokine induced by gamma interferon (MIG/CXCL9) has been shown to be expressed by IFN-gamma stimulated mononuclear cells and to attract activated T-cells through the chemokine receptor CXCR3. Since MIG is induced early in the response to IFN-gamma, measuring MIG may provide an interesting marker to assess downstream IFN-gamma induced responses, in contrast to assays that mainly focus on quantifying production of IFN-gamma per se. We, therefore, investigated MIG and IFN-gamma responses to a fusion protein of ESAT-6 and CFP-10, and compared responses to the conserved mycobacterial antigen 85B (Ag85B) and purified protein derivative (PPD) of M. tuberculosis, in 29 BCG vaccine controls and 24 TB patients. IFN-gamma secreting cells were determined by ELISPOT, and MIG production was measured by ELISA and flow cytometry. Production of MIG in response to ESAT-6/CFP-10, Ag85B and PPD correlated overall with increased numbers of IFN-gamma secreting cells (r=0.55, P<0.0001). A significant increase was noted among patients compared to controls in the secretion of IFN-gamma and MIG following stimulation with ESAT-6/CFP-10 or PPD (P<0.05). Moreover, MIG intracellular expression was higher in TB patients compared to BCG vaccines (P<0.05) in response to ESAT-6/CFP-10 or PPD. We conclude that MIG production correlates significantly with enhanced T-cell IFN-gamma production induced by M. tuberculosis-specific antigens ESAT-6/CFP-10. These results point to MIG as a potential novel biomarker that may be helpful in assessing downstream responses induced by IFN-gamma in TB.     

Human host genetic factors in mycobacterial and Salmonella infection: lessons from single gene disorders in IL-12/IL-23-dependent signaling that affect innate and adaptive immunity

Interleukin (IL)-12/IL-23 signal transduction-deficient individuals with genetic defects in IL12RB1 or IL12B often suffer from unusual mycobacterial and Salmonella infections. Here we discuss recent questions that have arisen from clinical observations that cast doubt on the necessity of IL-12/IL-23 signaling in controlling infections with intracellular bacteria. Alternative IL-12/IL-23-dependent, interferon-gamma-independent pathways of immunity to intracellular bacteria are also discussed.     

Undecanesulfonate does not allosterically activate H+ uniport mediated by uncoupling protein-1 in brown adipose tissue mitochondria

Undecanesulfonate is transported by uncoupling protein-1. Its inability to induce H+ uniport with reconstituted uncoupling protein-1 supports fatty acid cycling hypothesis. Rial et al. [Rial, E., Aguirregoitia, E., Jimenez-Jimenez, J., & Ledesma, A. (2004). Alkylsulfonates activate the uncoupling protein UCP1: Implications for the transport mechanism. Biochimica et Biophysica Acta, 1608, 122-130], have challenged the fatty acid cycling by observing uncoupling of brown adipose tissue mitochondria due to undecanesulfonate, interpreted as allosteric activation of uncoupling protein-1. We have estimated undecanesulfonate effects after elimination of endogenous fatty acids by carnitine cycle in the presence or absence of bovine serum albumin. We show that the undecanesulfonate effect is partly due to fatty acid release from albumin when undecanesulfonate releases bound fatty acid and partly represents a non-specific uncoupling protein-independent acceleration of respiration, since it proceeds also in rat heart mitochondria lacking uncoupling protein-1 and membrane potential is not decreased upon addition of undecanesulfonate without albumin. When the net fatty acid-induced uncoupling was assayed, the addition of undecanesulfonate even slightly inhibited the uncoupled respiration. We conclude that undecanesulfonate does not allosterically activate uncoupling protein-1 and that fatty acid cycling cannot be excluded on a basis of its non-specific effects.