Epitope Detection in the Envelope of Intracellular Naked Orthopox Viruses and Identification of Encoding Genes

Monoclonal antibodies (MAbs) were generated against vaccinia virus, cowpox virus KR2 Brighton, monkeypox virus Copenhagen, or ectromelia virus. Pairwise epitope specificity studies by competition ELISAs identified 23 distinct antigenic sites in 19 different orthopox virus strains. Six epitopes were completely independent of each other, and 17 closely related antigenic sites formed three separate epitope complexes. As shown by immunogold electron microscopy (ELMI), all MAbs reacted with epitopes in the envelope of intracellular naked virus, 16 MAbs recognized proteins of 32, 30, 16 or 14 kDa in Western blotting (WB), and 9 MAbs neutralized virus infectivity. In rabbitpox virus (RPV) 18 epitopes were detected. A λgt11 expression library of RPV DNA was screened with the corresponding 18 MAbs. Fourteen recombinant bacteriophage clones (ph) were isolated. Cross-hybridizations of phage and RPV DNA demonstrated a reaction with the HindIII A, HindIII D, or HindIII H fragments, respectively. DNA of ph3D was related to the A25L gene, which corresponds to the A-type inclusion body gene of cowpox virus. Two phage clones contained sequences of the 14-kDa fusion protein gene (A27L gene). Ph1A contained nearly the entire 14-kDa gene encoding 4 neutralizing (neutr) and 2 nonneutr epitopes. Ph5, expressing only half of this gene product, encoded 1 nonneutr epitope. The fusion protein of vaccinia virus MVA was isolated by immune-affinity chromatography with a neutr. catching MAb. The protein formed hollow rods (ELMI) and the 6 antigenic sites that were present were identical to those expressed by Escherichia coli infected with ph1A. WB detection with a polyclonal hyperimmune serum detected protein bands of 54, 32, 30, 16, and 14 kDa. The catching MAb bound only to a 16-kDa band. The purified fusion protein induced neutralizing antibodies in mice and rabbits.     

Pharmacokinetics of ascorbic acid in man

Summary The pharmacokinetic characteristics of ascorbic acid have been investigated in normal adult volunteers, using a two-compartment open model. After oral administration in a normal gelatine capsule the bioavailability of ascorbic acid was 69%. It was 98% when a sustained-release preparation was given in an identical capsule. During daily oral intake of ascorbic acid 1 g in the sustained-release form blood levels reached the equilibrium state within 3 days. Daily doses of ascorbic acid 1 g resulted in tissue saturation in 7 days, with a profound change in the pharmacokinetics of the vitamin. The binding of ascorbic acid to plasma proteins was low (24%). Its significance for the pharmacokinetic behaviour of the drug is discussed.     

A native antibody-based mobility-shift technique (NAMOS-assay) to determine the stoichiometry of multiprotein complexes

Characterization of multiprotein complexes (MPCs) is an important step toward an integrative view of protein interaction networks and prerequisite for a molecular understanding of how a certain MPC functions. Here, we present a technique utilizing monoclonal subunit-specific antibodies for an electrophoretic immunoshift assay in Blue Native-gels (NAMOS-assay), which allows the determination of the stoichiometry of MPCs. First, we use the B cell antigen receptor as a model MPC whose stoichiometry is known, confirming the HC(2)LC(2)Igalpha/beta(1) stoichiometry. Second, we demonstrate that the digitonin-extracted T cell antigen receptor (TCR) extracted from T cells has a stoichiometry of alphabetaepsilon(2)gammadeltazeta(2). We then show that the NAMOS-assay does not require purified MPCs, since it can determine the stoichiometry of an MPC in cell lysates. The NAMOS-assay is also compatible with use of epitope tags appended to the protein of interest, as e.g. the widely used HA-tag, and anti-epitope antibodies for the assay. Given its general applicability, this method has a wide potential for MPC research.     

Studies on the mechanism of paracetamol-induced protection against paracetamol hepatotoxicity.

In rats, 3 days treatment with paracetamol (1 oral dose of 1 g/kg daily) produced a complete protection against the hepatotoxic actions of a further dose of paracetamol as documented by determination of serum enzyme activities (glutamic-oxaloacetic transaminase, (GOT), glutamic-pyruvic transaminase (GPT), sorbitol dehydrogenase (SDH), bromsulphthalein retention and histological investigations. Subacute paracetamol treatment decreased liver glutathione levels by 46%, liver microsomal cytochrome P-450 content by 23%, hepatic hydroxylation of aniline by 29% and hepatic demethylation of aminopyrine by 46%. It afforded also some protection against the hepatotoxic actions of carbon tetrachloride, bromobenzene and thioacetamide, but did not influence the antiphlogistic activity of paracetamol (carrageenan paw edema test). Plasma and liver concentrations of free paracetamol after oral administration of 1 g/kg paracetamol were somewhat higher in the subacutely paracetamol-pretreated rats than in the non-pretreated control animals whereas no differences in the concentrations of conjugated paracetamol were found between the 2 groups. Pretreatment with paracetamol did not influence the urinary excretion of free paracetamol but caused some shift in the urinary excretion of paracetamol conjugates: pretreated rats excreted 23% less of the paracetamol glucuronide and sulfate and 33% more of the paracetamol mercapturate than the control animals. A depression of the microsomal mixed-function oxidase activity is presumed to be the main cause of the paracetamol-induced protection against paracetamol hepatotoxicity.     

Clinical prospective study of biochemical markers and evoked potentials for identifying adverse neurological outcome after thoracic and thoracoabdominal aortic aneurysm surgery

Background. Neurological deficit after repair of a thoracicor thoracoabdominal aortic aneurysm (TAA/TAAA) remains a devastatingcomplication. The aim of our study was to investigate the clinicalvalue of biochemical markers [S-100B, neurone-specific enolase(NSE) and lactate dehydrogenase (LD)], evoked potentials andtheir combinations for identifying adverse neurological outcomeafter TAA/TAAA surgery. Methods. From 69 patients, cerebrospinal fluid and blood samplesfor biochemical analysis were drawn after the induction of anaesthesia,during the cross-clamp period, 5 min, 2, 4, 6, 8, and 19 h,respectively, after reperfusion. In addition, continuous perioperativerecording of motor-evoked potentials after transcranial electricalstimulation (tcMEP) and somatosensory-evoked potentials wascarried out. Furthermore, neurological examinations were performed. Results. In patients with a defined decrease in lower extremitytcMEP during the cross-clamp period, we found that combinationsof the serum concentrations of S-100B and tcMEP ratios at 4,6, and 8 h after reperfusion had a positive and negative predictivevalue of 100% in predicting adverse neurological outcome afterTAA/TAAA surgery. Furthermore, combinations of the serum concentrationsof S-100B and NSE or LD at 19 h after reperfusion had both apositive and negative predictive value of 100% in identifyingpatients with adverse outcome after TAA/TAAA repair. Conclusions. TcMEP monitoring during TAA/TAAA surgery seemsto be an effective but not completely sufficient guide in ourprotective multi-modality strategy. Combinations of the serumconcentrations of S-100B and tcMEP ratios during the early reperfusionperiod might be associated with adverse neurological complications.Furthermore, biochemical markers could detect central nervoussystem injury on the first postoperative day and may have prognosticvalue.     

In vitro shear bond strength of self-etching adhesives in comparison to 4th and 5th generation adhesives

PURPOSE: To determine the shear bond strength (SBS) of different established (Resulcin Aqua Prime & Monobond N: RA, Prompt L-Pop III: PLP) and experimental (AC-Bond: AC, AC-Bond + Desensitizer: ACD) self-etching adhesives in comparison to fourth (Optibond FL: FL) and fifth generation (Excite: EX, Gluma Comfort Bond: CB) adhesives. MATERIALS AND METHODS: All adhesives were applied on flat enamel and dentin surfaces and light cured following manufacturers' directions. Tetric Ceram A2 composite cylinders 3.5 mm in diameter and 2.0 mm in height were sheared off (1 mm/min) after thermocycling (5 to 55 degrees C, 5000x). The t-test (5% level, Bonferroni-correction) was used for statistical analysis. RESULTS: SBS in enamel: RA: 27.0+/-5.8 MPa, PLP: 15.9+/-3.4 MPa, AC: 28.1+/-4.4 MPa, ACD: 22.2+/-4.1 MPa, FL: 33.2+/-3.2 MPa, EX: 30.5+/-5.1 MPa, CB: 30.1+/-3.7 MPa. SBS in dentin: RA: 25.8+/-5.7 MPa, PLP: 20.7+/-2.9 MPa, AC: 27.0+/-4.5 MPa, ACD: 20.7+/-3.7 MPa, FL: 34.4+/-3.8 MPa, EX: 30.0+/-4.6 MPa, CB: 27.9+/-2.6 MPa. FL resulted in significantly (p < 0.002) higher SBS in enamel and dentin than RA, AC, ACD, and PLP, and in higher SBS to dentin than CB. In enamel and dentin, RA performed significantly superior to PLP, but was not different from AC and ACD. EX and CB were both on the same level of significance as AC and RA, but showed superior results to ACD and PLP (enamel and dentin). PLP resulted in significantly lower SBS values in enamel and dentin than all the other materials investigated, except ACD in dentin, to which it was equivalent. CONCLUSION: Resulcin Aqua Prime & Monobond N and AC-Bond were not significantly different than established 5th generation products. AC-Bond + Desensitizer and Prompt L-Pop have significantly different SBS from established 4th and 5th generation products. Future studies are required to investigate marginal integrity to determine if self-etching adhesives are an adequate alternative to one- and multi-bottle systems.     

Modifying cochlear implant design: advantages of placing a return electrode in the modiolus.

HYPOTHESIS: A modiolar return electrode significantly increases the current flow across spiral ganglion cells into the modiolus, and may decrease the cochlear implant's power requirements. BACKGROUND: Ideal cochlear implants should maximize current flow into the modiolus to stimulate auditory neurons. Previous efforts to facilitate current flow through the modiolus included the fabrication and use of precurved electrodes designed to "hug" the modiolus and silastic positioners designed to place the electrodes closer to the modiolus. In contrast to earlier efforts, this study explores the effects of return electrode placement on current distributions in the modiolus. METHODS: The effects of return electrode positioning on current flow in the modiolus were studied in a Plexiglas model of the cochlea. Results of model measurements were confirmed by measurements in the modiolus of human temporal bones. The return electrode was placed either within the modiolus, or remotely, outside the temporal bone, simulating contemporary cochlear implant configurations using monopolar stimulation. RESULTS: Cochlear model results clearly show that modiolar current amplitudes can be influenced significantly by the location of the return electrode, being larger when placed into the modiolus. Temporal bone data show similar findings. Voltages recorded in the modiolus are, on average, 2.8 times higher with the return electrode in the modiolus compared with return electrode locations outside the temporal bone. CONCLUSION: Placing a cochlear implant's return electrode in the modiolus should significantly reduce its power consumption. Reducing power requirements should lead to improved efficiency, safer long-term use, and longer device life.