Magnetic biosensor technologies for medical applications: a review.

In this review we discuss conventional methods of performing biological assays and molecular identification and highlight their advantages and limitations. An alternative approach based on magnetic nanotechnology is then presented. Firstly, magnetic carriers are introduced and their biocompatibility and functionalisation discussed, with spotlights on functionalisation via self assembled monolayers and on methods of reducing nonspecific binding. In addition an introduction is provided to the basic physical concepts behind the various types of sensors used to detect magnetic labels. Finally, progress in the field of magnetic biosensors and the outlook for the future are discussed.

Detection of gamma-ray emission from the Vela pulsar wind nebula with AGILE.

Pulsars are known to power winds of relativistic particles that can produce bright nebulae by interacting with the surrounding medium. These pulsar wind nebulae are observed by their radio, optical, and x-ray emissions, and in some cases also at TeV (teraelectron volt) energies, but the lack of information in the gamma-ray band precludes drawing a comprehensive multiwavelength picture of their phenomenology and emission mechanisms. Using data from the AGILE satellite, we detected the Vela pulsar wind nebula in the energy range from 100 MeV to 3 GeV. This result constrains the particle population responsible for the GeV emission and establishes a class of gamma-ray emitters that could account for a fraction of the unidentified galactic gamma-ray sources.

Factors influencing theoretical knowledge and practical skill acquisition in student nurses: an empirical experiment.

A previous qualitative study [Nurse Education Today 20 (2000) 499] investigated perceptions of nurse teachers, student nurses and preceptors of the theory-practice gap said to exist within nursing. One theme was views of how the theory-practice gap could be closed. A subsequent quantitative study is reported here, in which this theme was translated into three factors. A full factorial experimental design was used to study the effect of these factors on theoretical knowledge and practical skill acquisition in a sample of first year undergraduate student nurses from one institution of higher education (n=19). The effect of whether a nurse teacher or preceptor taught students theoretical elements relating to a clinical specialty, whether the nurse teacher and preceptor collaborated on the content of what was taught to students and whether students went straight to, or delayed the clinical specialty following theoretical input, was examined. The results demonstrated preceptors were more effective than nurse teachers in promoting theoretical knowledge relating to their clinical specialty. Collaboration between the preceptors and nurse teachers on teaching content was ineffective at increasing theoretical knowledge. Delay between theoretical input and clinical experience was not detrimental for medical placements and for rehabilitation placements, resulted in an improved theoretical knowledge.

Mapping photogenerated radicals in thin polymer films: fluorescence imaging using a prefluorescent radical probe.

Prefluorescent radical probes, in which fluorescence is activated by radical trapping, and photoinitiators were used to detect radical generation in polymer films using fluorescence spectroscopy and microscopy. Prefluorescent radical probes are the foundation of a fluorescence imaging system for polymer films, that may serve both as a mechanistic tool in the study of photoinitiated radical processes in polymer films and in the preparation of functional fluorescent images.

Changes to water repellence of soil caused by the growth of white-rot fungi: studies using a novel microcosm system.

A microcosm system is described which permits assessment of the progressive growth of filamentous fungi through soil. We report on its application to measure the effects of Coriolus versicolor and Phanerochaete chrysosporium upon the sorptivity and water repellence of a mineral soil, measured using a miniature infiltration device. Both fungal species caused moderate sub-critical repellence. Since the pore structure was unaffected, the repellence was probably due to hydrophobic substances of fungal origin. This is the first report of changes in soil repellence caused by the growth of potential xenobiotic bioremediating fungi. The potential consequences are discussed.

Localization of Bunyamwera bunyavirus G1 glycoprotein to the Golgi requires association with G2 but not with NSm.

The Bunyamwera bunyavirus (BUN) M RNA genome segment encodes three proteins, two glycoproteins termed G1 and G2 and a non-structural protein called NSm, in the form of a polyprotein precursor that is co-translationally cleaved to give the mature proteins. Indirect immunofluorescence experiments have shown that these proteins localize to the Golgi complex in BUN-infected cells. We have used a recombinant vaccinia virus (vTF7-3), which expresses bacteriophage T7 RNA polymerase, to drive the expression of plasmids containing either the entire BUN M segment cDNA or fragments that encode the G1, G2 and NSm proteins separately under control of the T7 promoter. After transfection of these plasmids into vTF7-3-infected cells, correctly sized and processed proteins were detected by immunoprecipitation with BUN-specific antibodies. Immunofluorescence experiments showed that G1, G2 and NSm localized to the Golgi when transiently expressed from the full-length cDNA. When G2 or NSm were expressed separately they also localized to the Golgi, but when G1 was expressed alone a staining pattern typical for the endoplasmic reticulum was obtained. However coexpression of G2 and G1 from independent plasmids resulted in G1 localizing to the Golgi. In contrast translocation of G1 to the Golgi was not observed when G1 was coexpressed with NSm, although NSm itself was still detected in the Golgi. Similar results were obtained when the proteins were expressed from transfected plasmids containing the G2-, NSm- or G1-coding sequences under control of the cytomegalovirus immediate-early promoter. The localization of G1 to the Golgi when coexpressed with G2 was confirmed by the loss of endoglycosidase H (endo H) sensitivity of G1 after approximately 60 min in a pulse-chase experiment; G1 remained sensitive to endo H when expressed either alone or in combination with NSm. These results suggest that G2 contains the Golgi targeting and/or retention signals and that G1 has to interact with this protein to localize to this cellular compartment.

Simple method for scanning immunoblots.

Scanning laser densitometry was performed on immuno- and dot blots developed on nitrocellulose by treatment of the nitrocellulose with xylene. This method permits the development of simple methods for recording results of immunoblots and of producing semi-quantitative assays from dot blots.

Comparative activity of LY146032 against anaerobic cocci.

Fifty strains of anaerobic gram-positive cocci were tested in vitro against benzylpenicillin, teicoplanin, vancomycin and LY146032. The organisms displayed a wide range of susceptibility to penicillin and the minimum inhibitory concentration for 11 of the strains was greater than or equal to 1 mg penicillin/L. The activity of teicoplanin exceeded that of vancomycin by a factor of two. The activity of LY 146032 varied in different culture media and was dramatically potentiated by the addition of a physiological concentration of calcium. Peptococci were, in general, more susceptible than peptostreptococci to penicillin and to LY146032 in the absence of added calcium.

Guidelines for the production of polypeptide specific antisera using small synthetic oligopeptides as immunogens.

The production of antisera to specific proteins using as immunogens short, synthetic oligopeptides corresponding in sequence to regions of the proteins is analysed. Of 103 oligopeptides used for this purpose and reported in the literature before the end of 1983 all those corresponding to N or C terminal sequences produced antisera reacting with the complete protein. Of 69 oligopeptides corresponding to internal sequences only 71% were successfully used to prepare antisera. An analysis of these 69 oligopeptides showed that peptides of less than 10 amino acids were unlikely to produce useful antisera and that the more hydrophilic peptides were marginally more useful than those less hydrophilic.

Identification of herpes simplex virus DNA sequences which encode a trans-acting polypeptide responsible for stimulation of immediate early transcription.

Herpes simplex virus (HSV) immediate early (IE) transcription is known to be stimulated by a structural component of the virion which interacts, either directly or indirectly, with specific regulatory sequences located far upstream from IE messenger RNA 5'-termini. The aim of the work described in this paper is the mapping and identification of the virion component. Cloned HSV DNA fragments derived from various parts of the genome were cotransfected into BHK cells together with chimaeric plasmids which contained the thymidine kinase gene under IE control. Stimulation of thymidine kinase synthesis was elicited by cloned EcoRIi (0.63 to 0.72 map units), BamHIf (0.64 to 0.69) or EcoRIb (0.72 to 0.87). Cloned BamHIf had the same specificity as the virion component, since it stimulated thymidine kinase expression only from chimaeric plasmids which contained functional IE-specific regulatory sequences. The effect of EcoRIb was not confined to plasmids with IE-specific regulatory regions, suggesting a more general stimulatory role for one or more of the polypeptides encoded by this fragment. A subclone containing a 2.7 X 10(3) base-pair fragment of BamHIf (pMC1) was active in the cotransfection assay, and the effect was abolished by an eight base-pair insertion into the middle of this fragment. The only polypeptide known to map entirely within the HSV genome region defined by pMC1 was identified as the major tegument species Vmw65. The results therefore suggest that Vmw65 is the virion component which trans-activates HSV IE transcription.

Characterisation of a herpes simplex virus type 1 mutant which has a temperature-sensitive defect in penetration of cells and assembly of capsids.

A herpes simplex virus type 1 (HSV-1) mutant, ts1204, which has a temperature-sensitive (ts) mutation located within genome map coordinates 0.318 to 0.324, close to but outside the coding sequences of the glycoprotein gB gene, has been characterised. Although this mutant adsorbed to the cell surface at the nonpermissive temperature (NPT), it failed to penetrate the cell membrane. As a consequence of this defect, high multiplicities of infection of ts1204 blocked subsequent infection of cells by wild-type HSV-1. By contrast, at the NPT, superinfection of cells with HSV-2 was not inhibited by prior infection with ts1204. The penetration defect could be overcome either by brief incubation of mutant virus-infected cells at the permissive temperature, or by treatment of the cells with polyethylene glycol, a compound which promotes fusion of membranes. Upon continued incubation of ts1204-infected cells at the NPT, low numbers of capsids were assembled. Although these capsids all had some internal structure, they did not contain DNA. Another mutant, ts1208, which lies in the same complementation group as ts1204, penetrated cells normally at the NPT, but like ts1204, had a defect in the formation of functional capsids. Evidence presented in this paper suggests that the gene in which the ts1204 and ts1208 lesions map encodes a structural polypeptide.

Identification of a herpes simplex virus type 1 polypeptide which is a component of the virus-induced ribonucleotide reductase.

We have characterized a temperature-sensitive mutant of herpes simplex virus type 1 (HSV-1), 17tsVP1207, that induces a thermolabile ribonucleotide reductase activity. This mutant was derived from the multiple mutant tsG. Fine-structure mapping studies showed that the defect in 17tsVP1207 lies within an 800 bp sequence between genome map coordinates 0.580 and 0.585 in the gene encoding a polypeptide of 140 000 mol. wt. (Vmw136, ICP6). Since the mutation in this polypeptide produced a temperature-sensitive ribonucleotide reductase activity, Vmw136 must be a component of the herpes simplex virus-induced ribonucleotide reductase. The mRNA of Vmw136 has a common 3' terminus with an mRNA encoding a 38 000 mol. wt. polypeptide (Vmw38). Although the polypeptide-coding sequences of these mRNAs do not overlap, monoclonal antibodies against Vmw136 immunoprecipitated Vmw38 as well as Vmw136 from wild-type HSV-1-infected cells. Our data do not exclude the possibility that Vmw38 is part of the ribonucleotide reductase complex but suggest that this species on its own is not responsible for the HSV-induced enzyme activity.

Characterization of the 92,000-dalton glycoprotein induced by herpes simplex virus type 2.

Evidence is presented showing that the 92,000-dalton glycoprotein (g92K) induced by herpes simplex virus (HSV) type 2 has properties distinct from those assigned to any other HSV glycoprotein. First, the carbohydrate composition and extent of sulfation differ from those of glycoproteins D and E. Second, two clonally unrelated monoclonal antibodies, AP1 and LP5, shown in this paper to specifically immunoprecipitate g92K, do not react with any of the known processed forms of glycoproteins B, C, D, and E. Third, by using HSV type 1/HSV type 2 intertypic recombinants and a simple radioimmunoassay, the target antigen of the two monoclonal antibodies was shown to map in the same region as g92K (0.846 to 0.924). Fourth, the intertypic recombinant R12-3 was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of infected cells to induce the HSV type 2 g92K and HSV type 1 gD and GE, whereas R12-1, which did not induce g92K, induced HSV-2 gE and an altered gD, providing genetic evidence that g92K is encoded, at least in part, by a different region of the genome from that encoding gD and gE.

Successful use of oligopeptides as immunogens in the preparation of antisera to immediate-early gene products of herpes simplex virus type 1.

Antisera to two herpes simplex virus-type 1 (HSV-1) immediate-early gene products ( IE12 and IE175 ) have been produced using oligopeptides, constructed on the basis of proposed DNA coding sequences, as immunogens. In both cases the synthetic peptides were linked to bovine serum albumin via an N-terminal tyrosine prior to immunization. Both the IE12 and the IE175 antisera reacted with their respective HSV-1 antigens and with antigens produced by the HSV-1 immediate-early mutant tsK but not with functionally equivalent antigens induced by HSV-2. IE12 induced by tsK was found to have an altered mobility with respect to the wild-type IE12 and its precipitation was accompanied by a second, minor, component of lower mobility. Revertants of tsK gave similar results. Labelling IE12 with a variety of amino acids indicated that the first of three possible initiation codons was used in its translation from mRNA. The results imply that the other initiation codons were not used. Wildtype IE12 is produced for at least 3 h after release of cycloheximide block and appears to be turned over rapidly.

Processing of herpes simplex virus type 1 glycoproteins: two-dimensional gel analysis using monoclonal antibodies.

The number of discrete species of glycoprotein induced by herpes simplex virus type 1 (HSV-1, strain 17 syn+) and their processing has been examined by a combination of immunoprecipitation with monoclonal antibodies and analysis of immune precipitates by two-dimensional (2D) gel electrophoresis. Seventeen different monoclonal antibodies directed against glycoproteins A/B, C, D and E were used. Polypeptides intermediate in the synthesis of gA/B, C and D were visualized and two early intermediates of glycoprotein A/B (pgA/B1 and pgA/B2), both mannose-containing, were identified. Comparison on 2D gels of polypeptides synthesized in vitro from HSV-1-infected cell mRNA with those synthesized in pulse-chase experiments in vivo has allowed identification of early precursors of pgA/B and pgD. Their apparent mol. wt. were 105000 and 47000 respectively. The 2D gel analysis of glycoproteins induced in HFL cells infected with 17 syn+ revealed a number of previously unreported glycoprotein species. One had mobility on both gradient and single concentration SDS-polyacrylamide gels similar to that of gC but was resolved from gC on non-equilibrium pH gradient gels. This glycoprotein was produced in relatively large amounts and was not precipitated by any of the monoclonal antibodies used in this study. The data suggest that this glycoprotein either has a polypeptide chain unrelated to those of glycoproteins A/B, C, D or E or alternatively is derived from one of them and modified in such a way as to mask or remove the antigenic sites with which the monoclonal antibodies interact. Sixteen other previously unreported glycoprotein species not obviously related, as judged by their electrophoretic mobility, to gA/B, C, D or E were also identified: two reacted with gA/B-specific monoclonal antibodies and five with glycoprotein C-specific monoclonal antibodies. The origin of the remaining nine new glycoproteins has still to be ascertained.

Sulphated glycoproteins induced by herpes simplex virus.

BHK cells infected with strain 17 syn+ (HSV-1) or HG52 (HSC-2) incorporated inorganic sulphate into polypeptides which co-migrated on SDS-polyacrylamide gels with virus-induced glycoproteins. The major sulphated glycoprotein was glycoprotein E. In addition, less-intense sulphated bands co-migrated with glycoprotein D and HSV-1 glycoprotein A/B/C. Sulphate label co-migrating with HSV-2 glycoprotein A/B/C was occasionally observed. We have investigated which sulphated polypeptides are excreted from infected cells. Major ones of apparent mol. wt. 32000, 34000 and 35000 were excreted from cells infected with syn+. In addition, polypeptides which migrated in the vicinity of glycoprotein D were often excreted from cells infected with either 17 syn+ or HG52. The 32K, 34K and 35K polypeptides were antigenically related to glycoprotein D and over 95% of the total amount synthesized was excreted. Analysis of intracellular sulphated polypeptides using intertypic recombinants mapped glycoprotein E to between 0.832 and 0.950 units of the HSV genome.

Glial fibrillary acidic protein (GFAP): purification from human fibrillary astrocytoma, development and validation of a radioimmunoassay for GFAP-like immunoactivity.

The extraction and purification of glial fibrillary acidic protein (GFAP) from human fibrillary cerebellar astrocytoma is described. Using an immunoperoxidase method, antisera raised to the protein showed specific staining of astrocytes in normal spinal cord and in tumours of astrocytic origin. A double antibody radioimmunoassay for GFAP in tissue extract was developed, the detection limit of the assay being 360 pg. Extracts of tissues other than brain or spinal cord did not cross-react significantly in the assay, neither did purified preparations of myelin basic and S-100 proteins. Levels of GFAP in normal CNS tissue were higest in spinal cord (1370 microgram/g wet weight) but a level of 3050 microgram/g wet weight was detected in a fibrillary astrocytoma.

Radioimmunoassay of serum myelin basic protein and its application to patients with cerebrovascular accident.

Myelin basic protein-like immunoactivity was measured in the serum of patients after cerebrovascular accident (CVA) using a double antibody radioimmunoassay for myelin basic protein with a detection limit of 3 ng/ml serum. For up to 6 days after ictus, serum myelin basic protein levels in patients with severe CVA and patients who died as a result of CVA were significantly greater than those in control patients, patients with moderate CVA and patients surviving CVA. All patients with serum myelin basic protein levels greater than the range found in control subjects subsequently died. Serial dilutions of positive sera suggested that the immunoactivity differs from authentic myelin basic protein and may represent breakdown products of the protein. Serum from some patients with a previous history of moderate CVA had myelin basic protein binding activity consistent with the presence of antibodies to the protein.