Elemental analysis of the Mycobacterium avium phagosome in Balb/c mouse macrophages

Using a hard X-ray microprobe, we showed recently that in unstimulated peritoneal macrophages from C57BL/6 mice, the phagosome of pathogenic mycobacteria (Mycobacterium tuberculosis and Mycobacterium avium) can accumulate iron. We expanded our studies to the M. avium infection of peritoneal macrophages of Balb/c mice that show a similar degree of M. tuberculosis and M. avium-related chronic disease, but a higher susceptibility towards other intracellular pathogens such as Listeria monocytogenes, Leishmania major, or Brucella abortus as compared to C57BL/6 mice. Similar to C57BL/6 macrophages, the iron concentration in Balb/c macrophages increased significantly after 24 h of infection. A significant increase of the chlorine and potassium concentrations was observed in the Balb/c phagosomes between 1 and 24 h, in contrast with macrophages from C57BL/6 mice. The absolute elemental concentrations of calcium and zinc were higher in the mycobacterial phagosomes of Balb/c mice. We hypothesize that a potassium channel is abundant in the phagosome in macrophages that may be related to microbiocidal killing, similar to the requirement of potassium channels for microbiocidal function in neutrophils.     

Effect of roughage level in a total mixed ration on feed intake, ruminal fermentation patterns and chewing activity of early-weaned calves with ad libitum access to grass hay

In this study, two total mixed rations (TMR, based on dry ingredients) consisting (per kg dry matter (DM)) of 300 or 400 g finely chopped hay mixture of grass and alfalfa (H30 versus H40) were compared concerning their effects on dry matter intake (DMI), ruminal fermentation patterns and chewing activity of early-weaned (8 weeks milk-fed) calves. Ten ruminally cannulated male German Holstein calves were randomly assigned to two dietary treatments (n = 5) and observed from an age of 8–15 weeks. One group received the H30 (11.3 MJ metabolizable energy (ME)/kg DM) and the other the H40 (10.7 MJ ME/kg DM) TMR. All calves received grass hay (9.0 MJ ME/kg DM) separately. Water, TMR and hay were offered ad libitum twice daily (08:00 and 16:00 h). Rumen fluid was collected via cannula at an age of 9, 11, 13 and 15 weeks, twice weekly just prior to as well as 1, 3, 5 and 7 h after morning feeding. Chewing activity was recorded by a special head collar. As the calves aged DMI increased rapidly congruent with the recommended range for weaned calves. Because of the differing energy supply, calves receiving the H30 TMR were heavier than calves receiving the H40 TMR (139 kg versus 123 kg, P=0.007). During the trial ruminal pH of all calves were within the target range (6.2 ± 0.5), indicating physiological ruminal fermentation patterns. Daily mean ruminal pH was uninfluenced by treatment, however at an age of 13 and 15 weeks H30 showed a higher short chain fatty acid (SCFA) level than H40 (P=0.098; P=0.036). At an age of 15 weeks H30 showed a critical decrease in ruminal pH (3 h after feeding: 5.7) corresponding to a higher ruminal SCFA concentration (148.2 mmol/L, P=0.007). Chewing activity was well developed at an early age due to an increasing DMI after weaning. At an age of 15 weeks chewing activity (per day: 613–743 min total chewing; 358–418 min rumination) was similar to that of adult cows. In summary, feeding a dry TMR consisting per kg DM of 300–400 g hay to early-weaned calves can be recommended for a successful calf rearing up to an age of 15 weeks.     

Association of polymorphisms in the chemokine receptor CX3CR1 gene with coronary artery disease

Two chemokine receptor CX3CR1 gene variants, V249I and T280M, have been implicated in coronary artery diseases (CAD). Currently no consistent effect has been revealed and their role in cardiovascular disease is still conflicting. In the present study the association of CX3CR1 genotypes with CAD and myocardial infarction (MI) was investigated in the Ludwigshafen Risk and Cardiovascular Health (LURIC) cohort, including 3316 individuals in whom cardiovascular disease angiographically has been defined or ruled out. Similarly to previous studies, the alleles I249 and M280 were in strong linkage disequilibrium and formed an I₂₄₉M₂₈₀ haplotype. However, there was no relationship between CX3CR1 genotypes or corresponding haplotypes and the prevalence of CAD or MI. Adjusted for classical risk factors (age, sex, hypertension, dyslipidemia, diabetes mellitus and smoking), the odds ratio (OR) of V249I for CAD was 0.95 (95% confidence interval (CI) = 0.78–1.15, p = 0.61). The OR of T280M for CAD was 0.83 (95% CI = 0.66–1.04, p = 0.11). Furthermore, CX3CR1 variants were not associated with C-reactive protein levels, age at onset of CAD, severity of CAD and MI. In conclusion, present data of LURIC do not support the hypothesis that common variants of the CX3CR1 gene are associated with the presence of CAD or MI.     

The role of single subunits of the DNA transport machinery of Thermus thermophilus HB27 in DNA binding and transport

Thermus thermophilus HB27 is well known for its extraordinary trait of high frequencies of natural transformation, which is considered a major mechanism of horizontal gene transfer. We show that the DNA translocator of T. thermophilus binds and transports DNA from members of all three domains. These results, together with the data obtained from genome comparisons, suggest that the DNA translocator of T. thermophilus has a major impact in adaptation of Thermus to thermal stress conditions and interdomain DNA transfer in extreme hot environments. DNA transport in T. thermophilus is mediated by a macromolecular transport machinery that consists of at least 16 subunits and spans the cytoplasmic membrane and the entire cell periphery. Here, we have addressed the role of single subunits in DNA binding and transport. PilQ is involved in DNA binding, ComEA, PilF and PilA4 are involved in transport of DNA through the outer membrane and PilM, PilN, PilO, PilA1–3, PilC and ComEC are essential for the transport of DNA through the thick cell wall layers and/or through the inner membrane. These data are discussed in the light of the subcellular localization of the proteins. A topological model for DNA transport across the cell wall is presented.     

Legume phylogeny and the evolution of a unique contractile apparatus that regulates phloem transport

Protein bodies called forisomes undergo Ca(2+)-dependent deformations to occlude sieve tubes reversibly, providing a unique regulatory mechanism of phloem transport. Because forisomes are known exclusively from the Papilionoideae (Leguminosae), the evolution of forisome function may have played a role in the rapid radiation of this huge taxon. The unexpected discovery of a papilionoid species lacking forisomes led us to evaluate a representative set of species covering 33 of the 36 legume tribes traditionally recognized. We found forisomes in Papilionoideae but not in Caesalpinioideae and Mimosoideae. Forisomes were absent from several species of the papilionoid tribe Galegeae. Forisomes with tail-like protrusions occurred less frequently than tailless ones; their distribution correlated with taxonomic units but not sharply enough to render forisome type a reliable criterion for classification. Thus, the distribution of forisome types appeared to reflect physiological variability in the pathways of forisome assembly rather than the evolution of forisome genes. On the other hand, Ca(2+)-dependent forisome deformation and sieve tube plugging occurred in Bobgunnia madagascariensis, a member of the swartzioid clade that presumably is the sister group of all other papilionoids, suggesting that forisomes and their unique mechanism of deformation are a synapomorphy of the Papilionoideae.     

Nonantibiotic Properties of Tetracyclines: Structural Basis for Inhibition of Secretory Phospholipase A2

Secretory phospholipase A₂ is involved in inflammatory processes and was previously shown to be inhibited by lipophilic tetracyclines such as minocycline (minoTc) and doxycycline. Lipophilic tetracyclines might be a new lead compound for the design of specific inhibitors of secretory phospholipase A₂, which play a crucial role in inflammatory processes. Our X-ray crystal structure analysis at 1.65 Å resolution of the minoTc complex of phospholipase A₂ (PLA₂) of the Indian cobra (Naja naja naja) is the first example of nonantibiotic tetracycline interactions with a protein. MinoTc interferes with the conformation of the active-site Ca²⁺-binding loop, preventing Ca²⁺ binding, and shields the active site from substrate entrance, resulting in inhibition of the enzyme. MinoTc binding to PLA₂ is dominated by hydrophobic interactions quite different from antibiotic recognition of tetracyclines by proteins or the ribosome. The affinity of minoTc for PLA₂ was determined by surface plasmon resonance, resulting in a dissociation constant K_d = 1.8 × 10^− 4 M.     

Polyinosinic-polycytidylic acid treatment of Friend retrovirus-infected mice improves functional properties of virus-specific T cells and prevents virus-induced disease

The induction of type I IFN is the most immediate host response to viral infections. Type I IFN has a direct antiviral activity mediated by antiviral enzymes, but it also modulates the function of cells of the adaptive immune system. Many viruses can suppress type I IFN production, and in retroviral infections, the initial type I IFN is weak. Thus, one strategy of immunotherapy in viral infection is the exogenous induction of type I IFN during acute viral infection by TLR ligands. Along these lines, the TLR3/MDA5 ligand polyinosinic-polycytidylic acid [poly(I:C)] has already been used to treat viral infections. However, the immunological mechanisms underlying this successful therapy have not been defined until now. In this study, the Friend retrovirus (FV) mouse model was used to investigate the mode of action of poly(I:C) in antiretroviral immunotherapy. Postexposure, poly(I:C) treatment of FV-infected mice resulted in a significant reduction in viral loads and protection from virus-induced leukemia. This effect was IFN dependent because type I IFN receptor-deficient mice could not be protected by poly(I:C). The poly(I:C)-induced IFN response resulted in the expression of antiviral enzymes, which suppressed FV replication. Also, the virus-specific T cell response was augmented. Interestingly, it did not enhance the number of virus-specific CD4(+) and CD8(+) T cells, but rather the functional properties of these cells, such as cytokine production and cytotoxic activity. The results demonstrate a direct antiviral and immunomodulatory effect of poly(I:C) and, therefore, suggests its potential for clinical treatment of retroviral infections.