Biomedical subjects

R Schroeder

Publications and source records attributed to R Schroeder.

At least 37 records · Page 2Linked to original sources

PubMed1999

Assaying RNA chaperone activity in vivo using a novel RNA folding trap.

In the absence of proteins, RNAs often misfold in vitro due to alternative base pairings which result from the molecule being trapped in inactive conformations. We identify an in vivo folding trap in the T4 phage td gene, caused by nine base pairs between a sequence element in the upstream exon of the td gene and another at the 3' end of the intron. During translation, the ribosome resolves this interaction; consequently the intron folds correctly and splicing occurs. The introduction of a stop codon upstream of this base pairing prevents resolution of the inactive structure so that splicing cannot proceed. We have used this folding trap to probe for RNA binding proteins which, when overexpressed, either resolve the misfolded structure or impede its formation in vivo. We distinguish between proteins which recognize the intron structure and those which bind non-specifically and apparently ignore the intron. The first class, e.g. Neurospora crassa CYT-18, can rescue the exonic trap and intron mutants which cause a structural defect. However, known RNA chaperones such as Escherichia coli StpA and S12 and the HIV protein NCp7, only resolve the exonic trap without suppressing intron mutations. Thus, this structural trap enables detection of RNA chaperone activity in vivo.

Bacterial Proteins↗

PubMed1999

Analysis of the cooperative thermal unfolding of the td intron of bacteriophage T4.

The thermal stability of folded transcripts of the td intron of bacteriophage T4 that carried up to three base substitutions was investigated by temperature gradient gel electrophoresis (TGGE) and UV melting. The unfolding of this autocatalytic group I intron is endothermic and entropically driven. Although the effects of mutations in base pairs follow in most cases the expected order G-C>A-U>G.U>A.C, the extent of global destabilization varies strongly according to the helix in which substitutions are located. Effects are more pronounced in the P7 helix which forms, together with the P3 helix, the central pseudoknot of group I introns. The stability of the tertiary fold was also monitored as a function of ionic concentration and of the nature of the ion. At low ionic strength, the stabilizing effect of divalent ions is independent of the nature of the ion. However, with increasing ionic concentration, stabilization is most pronounced for Mg2+and less for Mn2+with Ca2+having intermediate effects. Ammonium ions stabilize folding with a similar slope, but at concentrations about 400 times higher than divalent ions. The apparent enthalpic change associated with the tertiary structure thermal unfolding increases strongly with increasing concentrations of divalent ions. A similar increase is observed with the monovalent ammonium ions. However, in the presence of NH4+ions, the apparent enthalpy peaks at 2.0 M and decreases beyond.

Bacteriophage T4↗

PubMed1999

Influence of specific mutations on the thermal stability of the td group I intron in vitro and on its splicing efficiency in vivo: a comparative study.

Group I introns constitute excellent systems for analyzing the relationship between RNA tertiary folding and catalysis. Within a hierarchical framework interpretation of RNA folding, secondary structure motifs subtend RNA three-dimensional (3D) architecture. Thus, mutations in two-dimensional motifs are expected to have effects different from those disrupting 3D contacts. Using UV spectroscopy, we have studied the influence of nucleotide substitutions, in both secondary and tertiary structure elements, on the thermal stability of the tertiary folding of the bacteriophage T4 td group I intron. Further, we present a quantitative analysis of the relationship between the splicing efficiency in vivo and the stability of the intron structure as monitored by UV melting curves. We conclude that the stability of the tertiary structure of a group I intron as measured by UV melting is generally a good indication of its ability to splice in vivo.

Base Sequence↗

PubMed1999

StreptoTag: a novel method for the isolation of RNA-binding proteins.

We describe a fast and simple one-step affinity-purification method for the isolation of specific RNA-binding proteins. An in vitro-transcribed hybrid RNA consisting of an aptamer sequence with high binding specificity to the antibiotic streptomycin and a putative protein-binding RNA sequence is incubated with crude extract. After complex formation, the sample is applied to an affinity column containing streptomycin immobilized to Sepharose. The binding of the in vitro-assembled RNA-protein complex to streptomycin-Sepharose is mediated by the aptamer RNA and the specifically bound proteins are recovered from the affinity matrix by elution with the antibiotic. Employing two well-characterized RNA-protein interactions, we tested the performance of this new method. The spliceosomal U1A protein and the bacteriophage MS2 coat protein could be isolated via their appropriate RNA motif containing hybrid RNA from crude yeast extracts in high yield and purity after only one round of affinity purification. As the purification principle is independent of the extract source, this new affinity chromatography strategy that makes use of an in vitro-selected antibiotic-binding RNA as a tag, "StreptoTag," should be applicable to extracts from other organisms as well. Therefore, we propose StreptoTag to be a versatile tool for the isolation of unknown RNA-binding proteins.

Base Sequence↗

PubMed1999

The antibiotic viomycin as a model peptide for the origin of the co-evolution of RNA and proteins.

Viomycin is an RNA-binding peptide antibiotic which inhibits prokaryotic protein synthesis and group I intron self-splicing. This antibiotic enhances the activity of the ribozyme derived from the Neurospora crassa VS RNA, and at sub-inhibitory concentrations it induces the formation of group I intron oligomers. Here, we address the question whether viomycin exerts specificity in the promotion of RNA-RNA interactions. In an in vitro selection experiment we tested the ability of viomycin to specifically select molecules out of an RNA pool. Group I intron RNA was incubated with a pool of random sequence RNA, or with a pool of RNA molecules which had previously been enriched for viomycin-binding RNAs. Viomycin was added in order to select viomycin-binding RNAs and to guide their interaction with the intron RNA resulting in recombinant molecules. Viomycin was indeed capable of specifically selecting RNA molecules which contain viomycin-binding sites promoting recombination. These results suggest that small peptides are able to play the role of selector molecules in a putative 'RNA World' launching the co-evolution of RNA and proteins into an 'RNA-protein World'.

Anti-Bacterial Agents↗

PubMed1999

Evaluation of apomorphine HCl effects on reproductive endpoints: studies in male rats and dogs.

Apomorphine HCl is currently in phase III clinical trials for the treatment of erectile dysfunction. The potential for reproductive toxicity in males was assessed based on a 13-week rat study--a fertility study in male rats--and a 6-month study in dogs. The subcutaneous (s.c.) route was selected to simulate the sublingual route in humans. Dosages of apomorphine were 0.0 (vehicle), 0.8, 2, and 8 mg/kg/day in the 13-week study in rats (20/group), with 8 mg/kg/day used for only 9 weeks. In the fertility study, 24 males/group were cohabited with females, and doses were 0.0, 0.2, 0.8, and 2 mg/kg/day. Males were treated for 4 weeks prior to cohabitation and for 5 weeks throughout cohabitation. Organ weights, including testis and left epididymis, sperm count and morphology in the epididymis, and sperm motility in the vas deferens were evaluated. Male fertility index, and in females, the numbers of fetuses, implantation sites, and corpora lutea were counted. Male dogs (five/group) were dosed with 0.04, 0.1, or 0.4 mg/kg/day for 6 months. Epididymal and prostate weight, and testicular and epididymal histology were evaluated. Daily morbidity/mortality, weekly clinical observations, body weight, and food consumption were evaluated in all studies. No adverse effect was observed in any of the reproductive parameters in the studies. The NOEL for reproductive toxicity was approximately equal to 0.4 mg/kg/day in dogs and > or = 2 mg/kg/day in rats. These doses in rats and dogs correlated with plasma levels of approximately 240 and 130 ng/mL, and AUCs of 200 and 100 ng.h/mL, respectively. These levels suggest a safety margin for the evaluated male reproductive endpoints of at least 104 times based on the Cmax, and 44 times based on AUC of the clinical dose.

Animals↗

PubMed1999

Fatigue-induced changes in bioprosthetic heart valve three-dimensional geometry and the relation to tissue damage.

BACKGROUND AND AIM OF THE STUDY: In a previous study, we used magnetic resonance (MR) imaging to reconstruct, three-dimensionally, porcine bioprosthetic heart valve (PBHV) cusp geometry. Initial results using three valves indicated that accelerated testing induced changes in cuspal shape, including focal regions of high curvature. Since for thin-walled shell structures, such as the PBHV cusp, curvature changes can affect the stress distribution independently from changes to mechanical properties, shape changes might have adverse effects on PBHV durability. METHODS: The MR technique was applied to an expanded valve database to explore more fully shape change with fatigue. The spatial curvature distribution was compared across valves subjected to a range of accelerated test times. RESULTS: Results confirmed our initial findings that PBHV cusps undergo a continuous, non-recoverable deformation with accelerated testing. This deformation resulted in an increase in the portion of cuspal surface exhibiting high curvature values. In one cusp we mapped structural information obtained by small-angle light scattering back to the three-dimensional cuspal surface using an interpolation technique. Results from the mapped cusp demonstrated a strong spatial correlation between elevated curvatures and structural damage. CONCLUSIONS: The observed changes in cuspal shape accelerate PBHV damage due to an increase in flexural strains induced by an increase in curvature reversal during operation, rather than an increase in tension during closure.

Bioprosthesis↗

PubMed1998

Antibiotic inhibition of RNA catalysis: neomycin B binds to the catalytic core of the td group I intron displacing essential metal ions.

The aminoglycoside antibiotic neomycin B induces misreading of the genetic code during translation and inhibits several ribozymes. The self-splicing group I intron derived from the T4 phage thymidylate synthase (td) gene is one of these. Here we report how neomycin B binds to the intron RNA inhibiting splicing in vitro. Footprinting experiments identified two major regions of protection by neomycin B: one in the internal loop between the stems P4 and P5 and the other in the catalytic core close to the G-binding site. Mutational analyses defined the latter as the inhibitory site. Splicing inhibition is strongly dependent on pH and Mg2+ concentration, suggesting electrostatic interactions and competition with divalent metal ions. Fe2+-induced hydroxyl radical (Fe-OH.) cleavage of the RNA backbone was used to monitor neomycin-mediated changes in the proximity of the metal ions. Neomycin B protected several positions in the catalytic core from Fe-OH. cleavage, suggesting that metal ions are displaced in the presence of the antibiotic. Mutation of the bulged nucleotide in the P7 stem, a position which is strongly protected by neomycin B from Fe-OH. cleavage and which has been proposed to be involved in binding an essential metal ion, renders splicing resistant to neomycin. These results allowed the docking of neomycin to the core of the group I intron in the 3D model.

Anti-Bacterial Agents↗

PubMed1998

A ribosomal function is necessary for efficient splicing of the T4 phage thymidylate synthase intron in vivo.

Splicing of the group I intron of the T4 thymidylate synthase (td) gene was uncoupled from translation by introducing stop codons in the upstream exon. This resulted in severe splicing deficiency in vivo. Overexpression of a UGA suppressor tRNA partially rescued splicing, suggesting that this in vitro self-splicing intron requires translation for splicing in vivo. Inhibition of translation by the antibiotics chloramphenicol and spectinomycin also resulted in splicing deficiency. Ribosomal protein S12, a protein with RNA chaperone activity, and CYT-18, a protein that stabilizes the three-dimensional structure of group I introns, efficiently rescued the stop codon mutants. We identified a region in the upstream exon that interferes with splicing. Point mutations in this region efficiently alleviate the effect of a nonsense codon. We infer from these results that the ribosome acts as an RNA chaperone to facilitate proper folding of the intron.

Bacteriophage T4↗

PubMed1998

Visualizing metal-ion-binding sites in group I introns by iron(II)-mediated Fenton reactions.

BACKGROUND: Most catalytic RNAs depend on divalent metal ions for folding and catalysis. A thorough structure-function analysis of catalytic RNA therefore requires the identification of the metal-ion-binding sites. Here, we probed the binding sites using Fenton chemistry, which makes use of the ability of Fe2+ to functionally or structurally replace Mg2+ at ion-binding sites and to generate short-lived and highly reactive hydroxyl radicals that can cleave nucleic acid and protein backbones in spatial proximity of these ion-binding sites. RESULTS: Incubation of group I intron RNA with Fe2+, sodium ascorbate and hydrogen peroxide yields distinctly cleaved regions that occur only in the correctly folded RNA in the presence of Mg2+ and can be competed by additional Mg2+, suggesting that Fe2+ and Mg2+ interact with the same sites. Cleaved regions in the catalytic core are conserved for three different group I introns, and there is good correlation between metal-ion-binding sites determined using our method and those determined using other techniques. In a model of the T4 phage-derived td intron, cleaved regions separated in the secondary structure come together in three-dimensional space to form several metal-ion-binding pockets. CONCLUSIONS: In contrast to structural probing with Fe2+/EDTA, cleavage with Fe2+ detects metal-ion-binding sites located primarily in the inside of the RNA. Essentially all metal-ion-binding pockets detected are formed by tertiary structure elements. Using this method, we confirmed proposed metal-ion-binding sites and identified new ones in group I intron RNAs. This approach should allow the localization of metal-ion-binding sites in RNAs of interest.

Animals↗

PubMed1998

Neomycin B inhibits splicing of the td intron indirectly by interfering with translation and enhances missplicing in vivo.

The aminoglycoside antibiotic neomycin B inhibits translation in prokaryotes and interferes with RNA-protein interactions in HIV both in vivo and in vitro. Hitherto, inhibition of ribozyme catalysis has only been observed in vitro. We therefore monitored the activity of neomycin B and several other aminoglycoside antibiotics on splicing of the T4 phage thymidylate synthase (td) intron in vivo. All antibiotics tested inhibited splicing, even chloramphenicol, which does not inhibit splicing in vitro. Splicing of the td intron in vivo requires translation for proper folding of the pre-mRNA. In the absence of translation, two interactions between sequences in the upstream exon and the 5' and 3' splice sites trap the pre-mRNA in splicing-incompetent conformations. Their disruption by mutations rendered splicing less dependent on translation and also less sensitive to neomycin B. Intron splicing was affected by neither neomycin B nor gentamicin in Escherichia coli strains carrying antibiotic-resistance genes that modify the ribosomal RNA. Taken together, this demonstrates that in vivo splicing of td intron is not directly inhibited by aminoglycosides, but rather indirectly by their interference with translation. This was further confirmed by assaying splicing of the Tetrahymena group I intron, which is inserted in the E. coli 23 S rRNA and, thus, not translated. Furthermore, neomycin B, paromomycin, and streptomycin enhanced missplicing in antibiotic-sensitive strains. Missplicing is caused by an alternative structural element containing a cryptic 5' splice site, which serves as a substrate for the ribozyme. Our results demonstrate that aminoglycoside antibiotics display different effects on ribozymes in vivo and in vitro.

Animals↗

PubMed1998

Angiotensin II-mediated expression of p27Kip1 and induction of cellular hypertrophy in renal tubular cells depend on the generation of oxygen radicals.

BACKGROUND: Angiotensin II (Ang II) induces hypertrophy of cultured proximal tubular cells. We have previously demonstrated that this Ang II-mediated hypertrophy occurs in the G1-phase of the cell cycle and depends on the induction of p27Kip1, an inhibitor of G1-phase cyclin/cyclin-dependent kinase complexes. The present study was undertaken to investigate whether Ang II may stimulate superoxide anions (O2.) formation in cultured LLC-PK1 and cultured mouse proximal tubule (MCT) cells, and to gain further insight into a potential relationship between O2. and cell cycle regulation. METHODS: Reactive oxygen species were measured with the lucigenin method in intact cells. The effects of various inhibitors were tested on Ang II-induced O2. production. Cells were transiently transfected with phosphorothioate-modified rat p22phox antisense oligonucleotides to investigate the potential role of NAD(P)H oxidase. Expression of p22phox mRNA after Ang II-treatment was detected with Northern blots. Incorporation of [3H]leucine into de novo synthesized proteins was used as a parameter of cell hypertrophy. Expression of p27Kip1 was evaluated in cell lysates by Western blotting. RESULTS: Ang II stimulated the accumulation of O2. in tubular cells; however, an addition of two different antioxidants completely abolished measurable O2. This effect was transduced by angiotensin receptor type-1 (AT1) and was inhibited by a flavoprotein inhibitor (DIP) or p22phox antisense oligonucleotides, indicating the involvement of membrane NAD(P)H oxidase. Ang II-stimulated de novo protein synthesis was attenuated by DIP, antioxidants, and p22phox antisense oligonucleotides. The Ang II-induced expression of p27Kip1 protein and cellular hypertrophy were reduced by similar treatments. Generation of O2. by xanthine supplementation also stimulated p27Kip1 expression and induced hypertrophy in LLC-PK1 cells. CONCLUSIONS: This study provides the first evidence, to our knowledge, that Ang II induces O2. in cultured tubular cells. Ang II-mediated activation of membrane bound NAD(P)H oxidase, probably by an increase in p22phox transcripts, is likely responsible for this induction. Generation of O2. subsequently induces p27Kip1 expression and stimulates hypertrophy, suggesting a novel mechanism of how Ang II can modulate cell cycle regulation.

Angiotensin II↗

PubMed1998

Assessment of vascularity in reactive lymph nodes by means of D-galactose contrast-enhanced Doppler sonography.

RATIONALE AND OBJECTIVES: A prospective study in signal-enhanced Doppler sonography of lymph nodes that were assumed pretherapeutically to be benign was performed to investigate characteristic sonomorphologic features and vascularity of reactively enlarged lymph nodes. METHODS: Thirty-four patients with enlarged superficial lymph nodes of the neck were examined first by B-scan sonography then by Doppler sonography before and after administration of an ultrasound signal-enhancing agent. In B-scan sonography, lymph nodes were classified into three groups according to their sonomorphologic features: (1) homogeneous parenchyma, (2) a centrally located echogenoic line, and (3) a echogenoic "hilus reflex." In conventional and contrast-enhanced Doppler sonography, peak flow rate, pulsatility index, and resistive index were assessed. Sonomorphologic criteria were compared with histologic findings. RESULTS: Reactively enlarged lymph nodes showed characteristic sonomorphologic patterns correlating to their histologic features. Echogenicity of the hilus corresponded to fibrosis (centrally located echogenoic line in 13 nodes; 38.2%) or fatty involution of the hili (echogenoic hilus reflex in 15 nodes; 44.1%). Administration of the galactose-based ultrasound contrast enhancer facilitated the assessment of hilar vessels, which projected to the echogenoic hili, respectively, actually additionally visualized a hilar vascularity in 10 of the 34 lymph nodes compared with conventional Doppler. Measured Doppler indices gave not a significant clue for identifying reactive lymph nodes or for differential diagnosis. CONCLUSIONS: Qualitative sonomorphologic assessment of characteristic sonomorphologic features of reactive lymph nodes may serve as a valuable tool for examining reactively enlarged lymph nodes. Administration of an ultrasound echo enhancer allows the assessment of a characteristic nodal vascularity in reactive lymph nodes and were superior to conventional B-mode and conventional Doppler sonography.

Adult↗

PubMed1998

Efficacy of CI-1020, an endothelin-A receptor antagonist, in hypoxic pulmonary hypertension.

We previously showed that CI-1020, an endothelin (ET)-A-selective receptor antagonist, dose-dependently blocked acute hypoxic pulmonary hypertension (PH) in rats. In this study we show that CI-1020 can reverse existing PH and prevent progression of right ventricular hypertrophy (RVH) in rats exposed to chronic hypoxia. Male Sprague-Dawley rats were exposed to 20 days of hypoxia (10% O2) with CI-1020 treatment (20 or 40 mg/kg/day) starting on day 10. On day 20 of hypoxia, the rats were instrumented under anesthesia with a pulmonary artery cannula and allowed to recover to consciousness before measurement of mean pulmonary arterial pressure (MPAP). Blood samples were then collected for plasma ET-1 measurements, the rats killed, and their hearts dissected, dried, and weighed. RV/LV + septum ratio (g/g) was used as an index of RVH (RVHi). Normoxic rats and rats exposed to hypoxia for only 10 days were also evaluated as controls. Normoxic rats had MPAPs of 13 +/- 1 mm Hg, plasma ET-1 levels of 2.1 +/- 0.1 pg/ml, and an average RVHi of 0.29 +/- 0.03. Rats exposed to 10 or 20 days of hypoxia had MPAPs of 33 +/- 2 and 44 +/- 0 mm Hg, plasma ET-1 levels of 4.2 +/- 0.8 and 4.6 +/- 0.8 pg/ml, and average RVHis of 0.47 +/- 0.05 and 0.52 +/- 0.03, respectively. In comparison, rats treated with CI-1020 had MPAPs that were 37% (20 mg/kg/day) and 44% (40 mg/kg/day) lower than untreated 20-day hypoxic rats. Furthermore, rats dosed with 40 mg/kg/day of CI-1020 had MPAPs that were significantly lower (24%) than control 10-day hypoxic rats, indicating a significant reversal of PH. Along with this reversal in PH, their average RVHi was 23% lower (p < 0.05) relative to untreated 20-day hypoxic rats.

Animals↗

PubMed1998

Glomerular expression of p27Kip1 in diabetic db/db mouse: role of hyperglycemia.

Early diabetic nephropathy is characterized by glomerular hypertrophy. Previous studies in vitro have demonstrated that mesangial cells exposed to high glucose are arrested in the G1-phase of the cell cycle and express increased levels of the cyclin-dependent kinase inhibitor p27Kip1. The present study was performed to investigate the renal expression of p27Kip1 in db/db mice, a model of diabetes mellitus type II. Glomerular p27Kip1 protein, but not mRNA expression, was strongly enhanced in diabetic db/db mice compared with non-diabetic db/+ littermates. Immunohistochemical studies revealed that this stimulated expression was mainly restricted to the nuclei of mesangial cells and podocytes, but glomerular endothelial cells occasionally also stained positively. Quantification of p27Kip1 positive glomerular cells showed a significant increase of these cells in db/db mice compared with non-diabetic db/+ animals. Although tubular cells revealed a positive staining for p27Kip1 protein, there was no difference between db/+ and db/db mice. Immunoprecipitation experiments revealed that p27Kip1 protein associates with Cdk2 and Cdk4, but not with Cdk6. To test for the influence of hyperglycemia on cell cycle arrest and p27Kip1 expression, mesangial cells were isolated from db/+ and db/db mice. There was a similar basal proliferation when these cells were grown in normal glucose-containing medium (100 mg/dl). However, raising the glucose concentration to 275 to 450 mg/dl induced cell cycle arrest in db/+ as well as db/db mesangial cells. Increasing the medium osmolarity with D-mannitol failed to induce p27Kip1 expression in mesangial cells. Transfection of cells with p27Kip1 antisense, but not missense, phosphorothioate oligonucleotides facilitated cell cycle progression equally well in db/+ and db/db mesangial cells. Furthermore, p27Kip1 expression was comparable in both cell lines in normal glucose, but increased in high glucose medium. Our studies demonstrate that p27Kip1 expression is enhanced in diabetic db/db animals. This induction appears to be due to hyperglycemia. Expression of p27Kip1 may be important in cell cycle arrest and hypertrophy of mesangial cells during early diabetic nephropathy.

Animals↗

PubMed1998

In vitro selection and characterization of streptomycin-binding RNAs: recognition discrimination between antibiotics.

As pathogens continue to evade therapeutical drugs, a better understanding of the mode of action of antibiotics continues to have high importance. A growing body of evidence points to RNA as a crucial target for antibacterial and antiviral drugs. For example, the aminocyclitol antibiotic streptomycin interacts with the 16S ribosomal RNA and, in addition, inhibits group I intron splicing. To understand the mode of binding of streptomycin to RNA, we isolated small, streptomycin-binding RNA aptamers via in vitro selection. In addition, bluensomycin, a streptomycin analogue that does not inhibit splicing, was used in a counter-selection to obtain RNAs that bind streptomycin with high affinity and specificity. Although an RNA from the normal selection (motif 2) bound both antibiotics, an RNA from the counter-selection (motif 1) discriminated between streptomycin and bluensomycin by four orders of magnitude. The binding site of streptomycin on the RNAs was determined via chemical probing with dimethylsulfate and kethoxal. The minimal size required for drug binding was a 46- and a 41-mer RNA for motifs 1 and 2, respectively. Using Pb2+ cleavage in the presence and absence of streptomycin, a conformational change spanning the entire mapped sequence length of motif 1 was observed only when both streptomycin and Mg2+ were present. Both RNAs require Mg2+ for binding streptomycin.

Base Sequence↗

PubMed1997

Human herpesvirus-6 (HHV-6)-associated necrotizing encephalitis in Griscelli's syndrome.

We report a male caucasian German pediatric patient of no Arab or Mediterranean ancestry with virus associated CNS lesions in Griscelli's syndrome (GS; McKusick No. 214450). The boy presented with recurrent infections, and meningitis with subsequent progressive signs of increased intracranial pressure leading to death at 32 weeks of age. At autopsy, various sites of the CNS revealed necroses in gray and white matter. CNS histology revealed numerous and massive predominantly perivascular CD8 positive lymphohistiocytic infiltrates. These findings were associated strictly with the presence of human herpesvirus-6 (HHV-6) genome or the HHV-6 specific late antigen H-AR 3, found in neurons, oligodendrocytes, and astrocytes. The search for HHV-6 replication dependent antigen, HHV-7 DNA, CMV, adenovirus, Coxsackie B1, B2, and B4-antigens, and mycobacteria was not successful. Detection of viruses was attempted using immunohistochemistry, in situ hybridization or nested polymerase chain reaction, respectively. Lymphocyte typing was carried out immunohistochemically. In GS, virus induced CNS damage does not seem to require necessarily active virus replication. It may also appear as a consequence of an immune reaction triggered by antigen expression.

Antigens, Viral↗

PubMed1997

Three-dimensional reconstruction from reduced sets of very noisy images acquired following a single-axis tilt schema: application of a new three-dimensional reconstruction algorithm and objective comparison with weighted backprojection.

In this work we propose a reconstruction algorithm (ART with blobs) that has not been previously used in electron Tomography and we compare it with the standard method in the field (weighted back projection, WBP). We assume that only a limited set of very noisy images, collected around a single axis tilt, is available; which is a typical situation in Electron Tomography. In general, the reconstruction problem is underdetermined (due to the limited number of projections) and the data are inconsistent (due to the high level of noise). The evaluation of the results is performed in a rigorous way by a task-oriented approach which makes use of numerical observers. ART with blobs outperforms WBP for a number of key tasks. Results are presented both for simplified line integral data and for realistic simulations of macromolecular structures embedded in amorphous ice.

Algorithms↗