Fowlpox virus (FPV) insertion plasmids were constructed that, upon integration into the viral genome via in-vivo recombination, inactivate the viral thymidine kinase (tk) gene. Using this approach, ...no wild-type virus-free stocks of recombinant virus could be obtained. In contrast, either integration of foreign genes into the intergenic region of the intact FPV tk gene and the open reading frame located downstream, or the functional substitution of the inactivated FPV tk gene by an intact vaccinia virus tk gene resulted in the predicted stable recombinants that were free of wild-type virus. Our results suggest that in already highly attenuated poxvirus strains an intact tk gene is essential for efficient growth of the virus in cell culture.
IMMUNO AG Biomedical Research Centre, 2304 Orth/Donau, Uferstrasse 15, Austria
Vaccinia virus is one of the most widely applied expression systems for use in eukaryotes in molecular biology. ...Expression of heterologous genes in the vaccinia virus system, however, requires integration of the foreign DNA into the vaccinia virus genome by means of homologous recombination or by direct molecular cloning. In both cases, plasmid vector constructs are required that contain the gene of interest and, usually, a marker gene, both of which are controlled by suitable promoter sequences. In order to simplify the construction of recombinants and to eliminate the need for a marker gene we have developed a modified vaccinia virus genome that allows the direct targeted insertion of DNA fragments downstream of a strong vaccinia virus promoter without any further cloning steps. The gene of interest is amplified by PCR using oligonucleotide primers that provide an Sfi I site at the 5' end and an Rsr II site at the 3' end of the PCR product. Following digestion with these restriction enzymes, the PCR product is operationally linked to a synthetic early/late promoter within the viral genomic DNA via the unique Sfi I/ Rsr II sites of the modified vaccinia virus genome. Using this approach, intermediate plasmid constructs and bacterial hosts are not required and time consuming screening steps can be omitted, because 90% of the virus progeny carry the foreign DNA.
* Author for correspondence. Fax +43 2212 2716.
Received 24 May 1995;
accepted 30 August 1995.
Physician response to a change in Medicaid fees McKay, N L; Dorner, F H
Health services management research : an official journal of the Association of University Programs in Health Administration,
05/1999, Letnik:
12, Številka:
2
Journal Article
Recenzirano
This study examines the effects of a change in Medicaid fees on the volume of physician services provided to beneficiaries. The data set includes price and volume at the procedure-level for Medicaid ...physician services in Texas in 1991, 1993, and 1995. The empirical analysis compares the volume of services provided to Medicaid participants before and after a 1992 change in reimbursement method. The results indicate that, over the period 1991 to 1993, the change in Texas Medicaid physician fees did not have a statistically significant effect on the volume of services provided. When measured over a longer period of time (1991-1995), however, volume increased significantly when price decreased, but, when price increased, there was no significant effect on volume. The results thus provide empirical support for the behavioural offset assumption underlying the switch to Medicare's Resource-Based Relative Value Scale (RBRVS) method of physician payment. A key policy implication is that reduced fees did not lead to a lower volume of physician services provided to Medicaid patients at least over the period of analysis. However, the new Medicaid fee schedule did not have the desired effect of controlling Medicaid expenditures on physician services.
Serum from rodents and felines has been found very effective in complement-dependent lysis of HIV-1, even in nonimmunized animals, but the effector molecules in animal serum and target structures on ...HIV-1 envelope gp120/160 responsible for complement activation were not determined. We have found that the natural anti-carbohydrate-specific IgM antibodies present in baby rabbit serum were able to lyse effectively the CD4+ T cells coated with the whole virus or with a recombinant gp120/160, irrespectively of the virus strain or glycoprotein expression system. When the high mannose-type glycans on gp160 were enzymatically removed by endoglycosidase F or blocked with the specific lectins, the complement activation and subsequent cell lysis were abolished. IgM-depleted baby rabbit serum was not able to lyse the gp120/160- and/or whole virus-coated target cells. These results suggest that the target structures for complement-activating and naturally occurring IgM antibodies in baby rabbit serum are high-mannose residues on HIV-1 envelope glycoprotein.
A sensitive and reliable quantitative method has been developed for the detection and quantitation of hepatitis C virus (HCV) target sequences. In this procedure, termed "laser induced fluorescence ...PCR' (LIF-PCR), reverse transcriptase PCR (RT-PCR) is performed and the PCR products are detected and quantified by laser-induced fluorescence. Precise quantitation of the viral target sequences is accomplished by the use of two calibrators that are amplified by the same set of primers as the target template. A high degree of reliability is achieved by co-processing, co-amplification and co-detection of the calibrators, together with the nucleic acid to be determined. Genome equivalents of HCV containing biological samples, including samples from international test panels, were accurately quantitated with this procedure.
This study uses data on hospital closures to examine the relation between exit and inefficiency in an industry where for-profit, not-for-profit, and government firms coexist. The likelihood of ...hospital exit over the period 1986-91 is estimated as a function of hospital relative inefficiency, ownership type, and other factors, where hospital relative inefficiency is measured using residuals from estimation of a stochastic frontier cost function. We find that less efficient hospitals were more likely to exit when ownership was for-profit or not-for-profit, but that relative inefficiency did not have a significant effect on the probability of exit for government hospitals.
A set of ten monoclonal antibodies (mabs) specific for the tick-borne encephalitis (TBE) virus envelope protein E were prepared and characterized with respect to their functional activities, the ...location of their binding sites on protein E and the involvement of their epitopes in acid pH-induced conformational changes and interactions with the precursor to the membrane protein (prM) in immature virions. The majority of these mabs mapped to the previously defined antigenic domain A. All of the mabs recognize parts of the E protein which undergo low pH-induced structural rearrangements believed to be necessary for the fusion activity of the virus, and six of the mabs define epitopes which are affected by the prM-E interaction in immature virions. They are therefore of potential value as specific reagents for studying the structure and function of protein E, as well as the function of the prM-E association. Five of the mabs exhibited neutralizing activity, and can therefore be used for the selection of escape mutants.
A study of the distances between physicians' (n=2,612) residency program locations and first practice locations indicates that most moved less than 75 miles. Primary care physicians moved ...significantly shorter distances than did other specialists. This pattern has changed little over the last few decades. (Author/MSE)
Foreign DNA was inserted into unique restriction endonuclease cleavage sites (Sma I or Not I) of the 200,000-base-pair vaccinia virus genome by direct molecular cloning. The modified vaccinia virus ...DNA was packaged in fowlpox virus-infected avian cells, and chimeric vaccinia virus was isolated from mammalian cells not supporting the growth of the fowlpox helper virus. In contrast to the classical "in vivo" recombination technique, chimeric viruses with inserts in both possible orientations and families of chimeras with multiple inserts were obtained. The different genomic configurations of chimeric viruses provide a broader basis for screening of optimal viruses. In addition to packaging in avian cells, a second packaging procedure for vaccinia DNA, based on the abortive infection of mammalian cells with the fowlpox helper virus, was developed. This procedure permits simultaneous packaging and host-range selection for the packaged virus. The cloning/packaging procedure allows the direct insertion of foreign DNA without the need for plasmids having flanking regions homologous to viral nonessential regions and is independent of inefficient in vivo recombination events. By direct cloning and packaging, about 5-10% of the total vaccinia virus yield consisted of chimeras. The procedure is, therefore, a useful tool in molecular virology.
A sensitive and reliable quantitative method based on the polymerase chain reaction (PCR) and the reverse transcription polymerase chain reaction (RT-PCR) to detect and quantify human ...immunodeficiency virus (HIV-1) and hepatitis B virus (HBV), respectively, was developed. The samples are co-processed together with two internal standards (calibrators). The amplicons are separated on denaturing polyacrylamide gels and co-detected and quantitated by laser induced fluorescence. HIV-1 and HBV containing biological samples, including samples from international test panels, were accurately quantitated. The procedure has proven to be a valuable tool in the quality control of biologicals such as plasma products and may serve to monitor disease progression and response to antiviral therapy.
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