Trypsin-sensitive and lipid-containing sites of the macrophage extracellular matrix bind apolipoprotein A-I and participate in ABCA1-dependent cholesterol efflux

A unique property of the extracellular matrix of J774 and THP-1 cells has been identified, which contributes to the ability of these cells to promote cholesterol efflux. We demonstrate high level apolipoprotein (apo) A-I binding to macrophage cells (THP-1 and J774) and to their extracellular matrix (ECM). However, high level apoA-I binding is not observed on fibroblasts, HepG2 cells, or U937 cells (a macrophage cell line that does not efflux cholesterol to apoA-I or bind apoA-I on their respective ECM). Binding to the ECM of THP-1 or J774 macrophages depends on the presence of apoA-I C-terminal helices and is markedly reduced with a mutant lacking residues 187-243 (apoA-I Delta(187-243)), suggesting that the hydrophobic C terminus forms a hydrophobic interaction with the ECM. ApoA-I binding is lost upon trypsin treatment or with Triton X-100, a preparation method that de-lipidates the ECM. However, binding is recovered with re-lipidation, and is preserved with ECM prepared using cytochalasin B, which conserves the endogenous phospholipid levels of the ECM. We also demonstrate that specific cholesterol efflux to apoA-I is much reduced in cells released from their native ECM, but fully restored when ECM-depleted cells are added back to ECM in the presence of apoA-I. The apoA-I-mediated efflux is deficient in plated or suspension U937 macrophages, but is restored to high levels when the suspension U937 cells are reconstituted with the ECM of J774 cells. The ECM-dependent activity was much reduced in the presence of glyburide, indicating participation of ABCA1 (ATP-binding cassette transporter 1) in the efflux mechanism. These studies establish a novel binding site for apoA-I on the macrophage ECM that may function together with ABCA1 in promoting cholesterol efflux.     

Heteronuclear NMR studies of human serum apolipoprotein A-I. Part I. Secondary structure in lipid-mimetic solution

Class I aminoacyl-tRNA synthetases have been thought to be single polypeptide enzymes. However, the complete genome sequence of a hyper thermophile Aquifex aeolicus suggests that the gene for leucyl-tRNA synthetases (LeuRS) is probably split into two pieces (leuS and leuS'). In this research, each gene was separately cloned and overexpressed in Escherichia coli and the protein products were examined for LeuRS activity. Leucylation activity was detected only when both gene products coexisted. Gel filtration analysis showed that the active form of A. aeolicus LeuRS has a heterodimeric (alpha/beta type) quaternary structure that is unique among class I aminoacyl-tRNA synthetases.     

Novel polymorphisms in <Emphasis Type="Italic">HLA-DOA</Emphasis> and <Emphasis Type="Italic">HLA-DOB</Emphasis> in B-cell malignancies

In B cells, HLA-DO controls HLA-DM-mediated peptide loading on MHC class II molecules. We analyzed whether HLA-DO mutations are associated with autoimmune diseases characterized by an autoantibody component and with a linkage to HLA-DR or HLA-DQ. These diseases include systemic lupus erythematosus, rheumatoid arthritis, celiac disease, and Graves' disease. In addition, several B-cell leukemias were screened for mutations in HLA-DO. A limited number of polymorphisms in DOA and DOB were found, most of which are non-coding changes or result in a conserved amino acid change. A novel non-conserved Arg to Cys mutation in DOA was found in a patient suffering from chronic lymphocytic leukemia. Further analysis did not reveal any effect on the function of HLA-DO. We conclude that HLA-DO variants are not critically involved in the autoimmune diseases and B-cell leukemias studied here.     

Serum trace elements status of rabbits supplemented with Nigella sativa,vitamins C and E,and selenium against damage by N-methyl-N'-nitro-N-nitrosoguanidine

In this study, we investigated the effects of Nigella sativa, vitamins C and E, and selenium on the levels of trace elements in the serum of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG)-injected rabbits. The rabbits were separated into one control and three experimental groups, each consisting of eight rabbits. MNNG was administered to all rabbits at a dose of 20 mg/kg. Group A received a suspension of N. sativa, group B received a combination of vitamins C and E and selenium, and group C received MNNG without any additional treatment. Group D did not receive any treatment and acted as control. The concentrations of serum zinc, copper, and iron were determined for groups A, B, C, and D. The zinc levels were 155.3±25.8, 304.7±14.22, 117.2±27.9, and 87.0±8 μ/dL for groups A-D, respectively; copper was measured at 234.8±31.9, 214.3±14.2, 196.5±19.3, and 359.2±19.9 μ/dL and iron levels were 276.3±10.71, 260.8±7.15, 211.2±13.47, and 223.4±9.5 μ/dL, in the stated group order. There were statistically significant differences between groups (p<0.05). The results obtained in this work may be of use for monitoring and preventing the nocive effects of N-methyl-N′-nitro-N-nitrosoguanidine and similar carcinogens.     

A Review of Studies on the Transmission of Anaplasma phagocytophilum from Sheep: Implications for the Force of Infection in Endemic Cycles

We review the findings of a longitudinal study of transmission of the intracellular tick-borne bacterium Anaplasma phagocytophilum from sheep to Ixodes ricinus ticks under natural conditions of tick attachment in the UK. In this study, sheep-to-tick transmission efficiency varied in a quadratic relationship with the number of adult ticks that were feeding on the sheep. We raise the hypothesis that this relationship may be due to conflicting effects of the density of ticks on bacterial survival and target cell (neutrophil) fluxes at the tick-host interface: in the same sheep at the same time, resistance to ticks was progressively inhibited with increasing number of feeding adult ticks, and investigation of serological responses to tick antigens suggesting loss of resistance may be associated with polarisation of host Th1 to Th2 type responses to ticks. We also raise the hypothesis that these properties, with superimposed effects on tick survival, may mean that variation in tick density is an important causal factor of observed variations in the force of A. phagocytophilum infection amongst different geographic foci. This revised version was published online in July 2006 with corrections to the Cover Date.     

Glutathione S-transferase polymorphisms influence the level of oxidative DNA damage and antioxidant protection in humans

The fem-3 gene of Caenorhabditis elegans was employed to determine the mutation frequency as well as the nature of mutations induced by low earth orbit space radiation ambient to Space Shuttle flight STS-76. Recovered mutations were compared to those induced by accelerated iron ions generated by the AGS synchrotron accelerator at Brookhaven National Laboratory. For logistical reasons, dauer larvae were prepared at TCU, transported to either Kennedy Space Center or Brookhaven National Laboratory, flown in space or irradiated, returned to TCU and screened for mutants. A total of 25 fem-3 mutants were recovered after the shuttle flight and yielded a mutation frequency of 2.1x10(-5), roughly 3.3-fold higher than the spontaneous rate of 6.3x10(-6). Four of the mutations were homozygous inviable, suggesting that they were large deletions encompassing fem-3 as well as neighboring, essential genes. Southern blot analyses revealed that one of the 25 contained a polymorphism in fem-3, further evidence that space radiation can induce deletions. While no polymorphisms were detected among the iron ion-induced mutations, three of the 15 mutants were homozygous inviable, which is in keeping with previous observations that high LET iron particles generate deficiencies. These data provide evidence, albeit indirect, that an important mutagenic component of ambient space radiation is high LET charged particles such as iron ions.