The E3-11.6-kDa adenovirus death protein (ADP) is required for efficient cell death: characterization of cells infected with adp mutants

We have reported that an 11,600-Da nuclear membrane glycoprotein named adenovirus death protein (ADP), encoded by the E3 region, is required for the efficient death (lysis) of adenovirus (Ad)-infected cells. We postulated that ADP mediates the release of virions from cells at the conclusion of replication. Here we provide further characterization of cells infected by adp+ and adp- Ads. Using virus mutants with deletions in the individual E3 genes, we show that only mutants that lack ADP have small plaques that are slow to develop. Mutants in the adp gene replicated as well as wild-type Ad, but the cells lysed much more slowly. Cell lysis and viability were determined by plaque size, cell morphology, trypan blue exclusion, the release of lactate dehydrogenase, and the MTT assay for mitochondrial activity. ADP is required for efficient lysis of human A549, KB, 293, and MCF-7 cells. A549 cells infected with adp+ Ads began to die at 2-3 days postinfection and were dead by 6 days. With adp mutants, > 80% of cells remained viable for 5-6 days; when the medium was changed, > 80% of cells were viable after 7 days and 10-20% after 14 days. When the MTT assay was used, there was an increase in mitochondrial activity, suggesting that Ad infection stimulates respiratory metabolism. Nearly all nuclei from wild-type Adinfected cells lacked DAPI-stained DNA by 7 days, whereas with an adp mutant nearly all nuclei stained brightly after 15 days. Nuclei from adp mutant-infected cells were extremely swollen and full of virus, and appeared to have an intact nuclear membrane. Cells infected with wild-type Ad had many vacuoles and perhaps a disrupted nuclear membrane; they did not display features typical of apoptosis.     

Dysregulated cytokine expression in vivo in prediseased and diseased autoimmune-prone MRL mice

Macrophages (m?) from prediseased autoimmune-prone MRL/ + and MRL/lpr mice produce markedly decreased levels of IL-1 in vitro in response to LPS. In contrast, tissues from diseased MRL/lpr mice overexpress IL-1 in vivo. To determine whether IL-1 underproduction in the MRL strains is solely an in vitro phenomenon, we compared in vivo cytokine mRNA expression from prediseased age-matched MRL/ + and MRL/lpr mice to that from normal BALB/c and C3HeB/FeJ mice. Like m? in vitro, whole organ RNA from the spleen, liver, and kidney of MRL/ + and MRL/lpr mice showed down-regulation of IL-1 RNA following intraperitoneal injection of LPS. This abnormality in inducible IL-1 expression was present in all MRL mice, irrespective of disease stage or the presence of the lpr gene. On the other hand, only diseased MRL/lpr mice displayed elevated and constitutive expression of IL-1 in their livers and kidneys. We suggest that inducible expression is most indicative of the intrinsic, or genetic, capacity of cells to produce cytokine, whereas constitutive expression reflects extracellular disease-related inflammatory stimuli present only in the diseased MRL/lpr strains. By restricting our studies to prediseased MRL mice, we have tried to eliminate the effects of disease and to focus on the predisposing genetic background. The existence both in vitro and in vivo of a defect in inducible IL-1 expression by prediseased MRL mice suggests that the molecular abnormality underlying this defect may be a part of this predisposing background to autoimmunity.     

Chemiluminescent detection of mRNAs on northern blots with digoxigenin end-labeled oligonucleotides

To establish a simplified, nonradioactive approach for identifying mRNAs on Northern blots, antisense oligonucleotides have been used as probes in combination with chemiluminescence-based detection. Oligonucleotides (approximately 32-mer) were end-labeled with digoxigenin (DIG) and used in conjunction with adamantyl 1,2-dioxetane aryl phosphate substrates (Lumigen PPD and CSPD). Oligonucleotides were designed as probes for several mRNAs in tissues of rats and mice, including the mitochondrial uncoupling protein, lipoprotein lipase, GLUT1, GLUT4, and beta-actin. Uncoupling protein mRNA was detected in total RNA from brown adipose tissue with a 32-mer DIG-labeled oligonucleotide, within 2 min of exposure to film. This mRNA could also be detected when as little as 250 ng of total RNA was applied to the gel, following 4 h exposure to film, and was present only in brown fat. The mRNA for lipoprotein lipase was detectable with a 30-mer DIG-labeled oligonucleotide in 1 micrograms of total RNA from mouse heart, within 2 h of exposure. The mRNA for the GLUT1 glucose transporter was detected in total RNA from rat midbrain using a 32-mer DIG-labeled oligonucleotide, while beta-actin mRNA was detected with a 30-mer oligonucleotide. The mRNA for the insulin-sensitive glucose transporter GLUT4 was detected with a 32-mer DIG-labeled oligonucleotide and found only in those tissues in which glucose uptake is stimulated by insulin. The speed of detection was greater with CSPD and was augmented by exposure of membranes to film at 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

The mechanism of plantar unloading in total contact casts: implications for design and clinical use

Although the total contact cast (TCC) has been shown to be an extremely effective treatment for the healing of plantar ulcers in diabetic patients, little is known about the biomechanics of its action. In this study, plantar pressure and ground reaction force measurements were obtained from over 750 foot contacts as five subjects with known elevated plantar forefoot pressures walked barefoot, in a padded cast shoe, and a TCC. Peak plantar pressures in the forefoot were markedly reduced in the cast compared with both barefoot and shoe walking (reductions of 75% and 86% respectively, P < 0.05). Peak plantar pressures in the heel were not, however, significantly different between the shoe and the TCC, and the longer duration of heel loading resulted in an impulse that was more than twice as great in the cast compared with the shoe (P < 0.05). An analysis of load distribution indicated that the mechanisms by which the TCC achieves forefoot unloading are (1) transfer of approximately 30% of the load from the leg directly to the cast wall, (2) greater proportionate load sharing by the heel, and (3) removal of a load-bearing surface from the metatarsal heads because of the "cavity" created by the soft foam covering the forefoot. These results point out some of the "essential design features" of the TCC (which are different from what had been previously supposed), support the use of the TCC for healing plantar ulcers in the forefoot, but raise questions about its utility in the healing of plantar ulcers on the heel.     

Solution scattering structural analysis of the 70 S Escherichia coli ribosome by contrast variation. I. Invariants and validation of electron microscopy models

Solutions of selectively deuterated 70 S Escherichia coli ribosomes and of free 30 S and 50 S subunits were studied by neutron scattering using contrast variation. The integrity of the partially deuterated particles was controlled by parallel X-ray measurements. Integral parameters of the entire ribosome, of its subunits and of the protein and rRNA moieties were evaluated. The data allow an experimental validation of the two most recent electron microscopy reconstructions of the 70 S ribosome presented by the groups of J. Frank (Albany) and of M. van Heel & R. Brimacombe (Berlin). For each reconstruction, integral parameters and theoretical scattering curves from the 70 S and its subunits were calculated and compared with the experimental data. Although neither of the two models yields a comprehensive agreement with the experimental data, Frank's model provides a better fit. For the 50 S subunit of van Heel & Brimacombe's model the fit with the experimental data improves significantly when the internal channels and tunnels are filled up. The poorer fit of the latter model is thus caused by its "sponge"-like structure which may partly be due to an enhancement of high frequency contributions in some of the steps of the three-dimensional image reconstruction. It seems therefore unlikely that the ribosome has a "sponge"-like structure with a pronounced network of channels.     

Evaluation of extracellular lipid peroxidation in brain cortex of anaesthetized rats by microdialysis perfusion and high-performance liquid chromatography with fluorimetric detection

OBJECTIVE: In the context of the need to develop practical outcome measures, the present study aimed to assess the sensitivity of the Life Skills Profile (LSP) in terms of differences between hospital-based and community-based clients, and to assess the sensitivity of the LSP to changes over time. In this way, criteria could be established whereby the LSP could be used to determine appropriate changes in locus of care, both in terms of the "cut-off' for hospital-based and community-based tenure, and the level of "clinically significant change' in functioning. METHOD: The LSP was administered at 3-monthly intervals to 200 clients of an area public mental health service with serious mental illness over a 21-month period. Locus of care (hospital or community) was noted at each administration. RESULTS: Clients in the community scored significantly better than those in hospital, however there was a great deal of overlap. Using hospital or community tenure as the variable of interest, a measure of reliable and clinically significant change over a 3-month period based on the LSP was developed. A total LSP score of 116.5 or above best discriminated clients in the community from those in hospital, and a difference of 18 points or more in two LSP obtained 3 months apart was unlikely to have arisen by chance. A simple, two-part criterion of significant change based on these results showed 89% accuracy in matching transition (or lack of transition) between hospital and community with changes in LSP scores. CONCLUSIONS: The results need to be understood within the methodological limitations of the present study. The findings provide users of the LSP with guidelines for the interpretation of repeat assessments. This may encourage more services to use formal reassessment methods to monitor the progress of their clients.