Oligomerization of ICP4 and rearrangement of heat shock proteins may be important for herpes simplex virus type 1 prereplicative site formation

Herpes simplex virus type 1 (HSV-1) DNA replication occurs in replication compartments that form in the nucleus by an ordered process involving a series of protein scaffold intermediates. Following entry of viral genomes into the nucleus, nucleoprotein complexes containing ICP4 can be detected at a position adjacent to nuclear domain 10 (ND10)-like bodies. ND10s are then disrupted by the viral E3 ubiquitin ligase ICP0. We have previously reported that after the dissociation of ND10-like bodies, ICP8 could be observed in a diffuse staining pattern; however, using more sensitive staining methods, we now report that in addition to diffuse staining, ICP8 can be detected in tiny foci adjacent to ICP4 foci. ICP8 microfoci contain UL9 and components of the helicase-primase complex. HSV infection also results in the reorganization of the heat shock cognate protein 70 (Hsc70) and the 20S proteasome into virus-induced chaperone-enriched (VICE) domains. In this report we show that VICE domains are distinct but adjacent to the ICP4 nucleoprotein complexes and the ICP8 microfoci. In cells infected with an ICP4 mutant virus encoding a mutant protein that cannot oligomerize on DNA, ICP8 microfoci are not detected; however, VICE domains could still be formed. These results suggest that oligomerization of ICP4 on viral DNA may be essential for the formation of ICP8 microfoci but not for the reorganization of host cell chaperones into VICE domains.     

Parental transmission of <Emphasis Type="Italic">HLA-DRB1*15 </Emphasis>in multiple sclerosis

Chen et al. found that the CA haplotype of protein C -1654C/T and -1641G/A was associated with increased risk of death and organ dysfunction in Chinese Han patients with severe sepsis (Hum Genet 123:281–287, 2008). We similarly tested for association of the C allele of protein C 673 T/C (rs2069912) (linkage disequilibrium with the CA haplotype, D′ = 100%) in a cohort of 100 North American East Asians with severe sepsis. The C allele was associated with increased mortality and organ dysfunction, consistent with Chen et al. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Support: Sirius Genomics Inc., Canadian Institutes of Health Research. Keith R. Walley is a Michael Smith Foundation for Health Research Distinguished Scholar.     

Nitrogen mineralization and phenol accumulation along a fire chronosequence in northern Sweden

Scots pine (Pinus sylvestris L.) forests of northern Sweden are often considered to be N limited. This limitation may have been exacerbated by the elimination of wildfire as a natural disturbance factor in these boreal forests. Phenolic inhibition of N mineralization and nitrification (due to litter and exudates of ericaceous shrubs) has been proposed as a mechanism for N limitation of these forests, but this hypothesis remains largely untested. N mineralization rates, nitrification rates, and sorption of free phenolic compounds were assessed along a fire-induced chronosequence in northern Sweden. A total of 34 forest stands varying in age since the last fire were identified and characterized. Overstorey and understorey vegetative composition and depth of humus were analysed in replicated plots at all 34 sites. Eight of the forest stands aged 3-352 years since the last fire were selected for intensive investigation in which ten replicate ionic resin capsules (used to assess net N mineralization and nitrification) and non-ionic carbonaceous resin capsules (used to assess free phenolic compounds) were installed at the interface of humus and mineral soil. A highly significant correlation was observed between site age and net sorption of inorganic N to resin capsules. Net accumulation of NH₄⁺ and NO₃^- on resin capsules followed a linear decrease (R²=0.61, P<0.01) with time perhaps as a result of increased N immobilization with successional C loading. NO₃^- sorption to resin capsules followed a logarithmic decrease (R²=0.80, P<0.01) that may be related to a logarithmic increase in dwarf shrub cover and decreased soil charcoal sorption potential along this chronosequence. A replicated field study was conducted at one of the late successional field sites to assess the influence of charcoal and an added labile N source on N turnover. Three rates of charcoal (0, 100, and 1,000 g M^-2) and two rates of glycine (0 and 50 g N as glycine M^-2) were applied in a factorial design to microplots in a randomized complete block pattern. Net ammonification (as assessed by NH₄⁺ sorption to resins) was readily increased by the addition of a labile N source, but this increase in NH₄⁺ did not stimulate nitrification. Nitrification was stimulated slightly by the addition of charcoal resulting in similar levels of resin-sorbed NO₃^- as those found in early successional sites. Resin-sorbed polyphenol concentrations were decreased with charcoal amendments, but were actually increased with N amendments (likely due to decomposition of polyphenols). Net N mineralization appears to be limited by rapid NH₄⁺ immobilization whereas nitrification is limited by the lack of an appropriate environment or by the presence of inhibitory compounds in late successional forests of northern Sweden.     

Photoaffinity labeling of the 1,25-dihydroxyvitamin D-3 receptor.

Underivatized 1,25-dihydroxy[26,27-3H]vitamin D-3 was successfully used to photoaffinity label the 1,25-dihydroxyvitamin D-3 receptor. The covalent incorporation of tritium into the receptor protein was induced by ultraviolet irradiation of the receptor-1,25-dihydroxy[26,27-3H]vitamin D-3 complex in crude pig intestinal nuclear extract. The amount of incorporated label increased with increasing time of irradiation and was dependent on light of wavelengths 220-280 nm. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and fluorography were used to demonstrate that label was incorporated primarily into the 1,25-dihydroxyvitamin D-3 receptor. In addition, the label incorporation was eliminated by competition with a 100-fold excess nonradioactive 1,25-dihydroxyvitamin D-3, indicating that the label was specific for the steroid binding site. Since 1,25-(OH)2[26,27-3H]vitamin D-3 is readily available and requires no special precautions for its preparation and handling, it should be a useful photoaffinity label for future studies of the receptor.     

Stabilization of 1,25-dihydroxyvitamin D-3 receptor in the human leukemia cell line, HL-60, with diisopropylfluorophosphate

We have investigated the reason for the lack of specific 1,25-dihydroxyvitamin D-3 binding activity in extracts of ATCC HL-60 cells. Although intact ATCC HL-60 cells specifically and saturably take up 1,25-dihydroxy[3H]vitamin D-3, whole cell extracts have little or no specific binding of 1,25-dihydroxyvitamin D-3. The absence of specific binding can now be explained by the action of a serine proteinase in these cells. When diisopropylfluorophosphate (DFP), a potent inhibitor of serine proteinase, is added to the buffer used for extraction, specific binding of 1,25-dihydroxy[3H]vitamin D-3 in the extract is observed. The loss of specific binding could not be prevented by hydrolyzed DFP or other serine proteinase inhibitors, such as phenylmethylsulfonylfluoride, benzamidine and aprotinin. The proteolytic activity from ATCC cells also destroyed specific 1,25-dihydroxy[3H]vitamin D-3 binding in high-salt extracts from pig intestinal nuclei or from another HL-60 cell line (LG HL-60 cells). However, the proteinase did not affect the levels of the specific binding in these preparations if the receptor was occupied with 1,25-dihydroxy[3H]vitamin D-3 prior to exposure to the proteinase. The binding and sedimentation characteristics of the receptors from various sources were not changed by the presence of DFP. The Kd of the receptor in ATCC HL-60 cells is 1.2.10(-10) M, which is identical to that in the LG HL-60 cells. The 1,25-dihydroxy[3H]vitamin D-3 receptor complex from the ATCC cells sediments as a single 3.5 S component and elutes from DNA-Sephadex column in two peaks at 0.09 and 0.15 M KCl. The material eluting at 0.15 M KCl has the same DNA-binding activity as preparations from pig intestine or LG HL-60 cells. Immunoprecipitation studies demonstrated that monoclonal antibodies to the pig receptor, IVG8C11, quantitatively precipitate the 1,25-dihydroxy[3H]vitamin D-3-binding activity from ATCC HL-60 cells as well as that from LG HL-60 cells or pig intestinal nuclei. Therefore, the previous failure to demonstrate the 1,25-dihydroxyvitamin D-3 receptor in ATCC HL-60 cells is because of the presence of a potent serine proteinase and not because of an abnormal or absent receptor.