Physician education in hyperlipidemia management: the impact on collaboration.

Collaboration of health care professionals is likely beneficial in modifying patient behavior in the treatment of hyperlipidemia. The purpose of this study was to determine whether limited instruction and demonstration of collaborative management of hyperlipidemia in a continuing medical education (CME) would change physicians' office practices, as determined 1 year later by questionnaire. Collaborative practice was defined as physicians working with other allied health care professionals as a team to increase patients' medication compliance and other behavioral outcomes. A 19-credit hour CME Lipid Disorders Training Program (LDTP) was offered emphasizing the collaborative approach to hyperlipidemia patient management. Physicians (n = 196) were surveyed 1 year after LDTP. The response rate was 52.5%, nonrespondents were similar in locations. About 51% of respondents reported increased collaborative practice; of these respondents, 68% reported saving time, 78% reported improved patient outcomes, 76% improved office efficiency, and 90% increased patient satisfaction. According to self-reporting by these physicians, increased collaboration practices after attending the LDTP course led to improved patient outcomes.

Serological recognition of feline infectious peritonitis virus spike gene regions expressed as synthetic peptides and E. coli fusion protein.

Cats exposed to feline infectious peritonitis virus (FIPV) or feline enteric coronavirus (FECV) cannot be differentiated by serological analysis. Three synthetic peptides and an E. coli recombinant fusion protein generated from FIPV 79-1146 spike gene sequence were produced. Coronavirus positive cat sera reacted to peptide aa 950-990 but were non-reactive to aa137-151 and aa 150-180 peptides as demonstrated by ELISA. Amino acid sequence 97-222 expressed as a galk fusion protein in E. coli was tested against coronavirus positive cat sera by western blot analysis. Only sera from cats exposed to the FIPV type-II strains DF-2 or 79-1146 that were exhibiting signs of FIP recognized the fusion protein. Sera from FECV exposed cats did not recognize the 97-222 fusion protein in western blot analysis.

Characterization of rabies glycoprotein expressed in yeast.

The rabies virus surface glycoprotein was synthesized in Saccharomyces cerevisiae using an expression vector which contains an inducible promoter from the copper metallothionein gene. The rabies G protein was also expressed constitutively in yeast when cloned under control of the triose dehydrogenase promoter. Polypeptides of 65-68 kDa, which migrated at the same molecular weight as authentic viral rabies G protein species, were synthesized by yeast transformants as detected by immunoblotting with rabies specific antiserum. In addition, these polypeptides were immunoprecipitated with several rabies G-specific monoclonal antibodies which neutralize virus infectivity. The recombinant rabies G proteins were glycosylated and associated with membranes in yeast. When injected into guinea pigs, yeast extracts containing the rabies G protein protected animals from lethal rabies virus challenge when administered intramuscularly. However, the same material did not protect mice from a lethal rabies intracerebral challenge.

Identification, sequence, and expression of the gene encoding the second-largest subunit of the vaccinia virus DNA-dependent RNA polymerase.

The gene, rpo 132, encoding the second-largest subunit of the vaccinia virus DNA-dependent RNA polymerase was identified and sequenced. Two complementary approaches, involving antiserum to purified vaccinia virus RNA polymerase, were used to locate the rpo 132 gene. One method involved the screening of a lambda gt11 library of vaccinia virus genome fragments and the other was based on the immunoprecipitation and polyacrylamide gel electrophoresis of the in vitro translation products of mRNA that hybridized to immobilized vaccinia virus DNA. The deduced open reading frame of the rpo 132 gene predicted a polypeptide of 1164 amino acid residues with sequence similarities to the second-largest RNA polymerase subunits of eubacteria, archaebacteria, and eukaryotes as well as to other poxviruses. Transcriptional analyses indicated that rpo 132 has both early and late RNA start sites and is expressed throughout infection.

Identification of rpo30, a vaccinia virus RNA polymerase gene with structural similarity to a eucaryotic transcription elongation factor.

Eucaryotic transcription factors that stimulate RNA polymerase II by increasing the efficiency of elongation of specifically or randomly initiated RNA chains have been isolated and characterized. We have identified a 30-kilodalton (kDa) vaccinia virus-encoded protein with apparent homology to SII, a 34-kDa mammalian transcriptional elongation factor. In addition to amino acid sequence similarities, both proteins contain C-terminal putative zinc finger domains. Identification of the gene, rpo30, encoding the vaccinia virus protein was achieved by using antibody to the purified viral RNA polymerase for immunoprecipitation of the in vitro translation products of in vivo-synthesized early mRNA selected by hybridization to cloned DNA fragments of the viral genome. Western immunoblot analysis using antiserum made to the vaccinia rpo30 protein expressed in bacteria indicated that the 30-kDa protein remains associated with highly purified viral RNA polymerase. Thus, the vaccinia virus protein, unlike its eucaryotic homolog, is an integral RNA polymerase subunit rather than a readily separable transcription factor. Further studies showed that the expression of rpo30 is regulated by dual early and later promoters.

Identification of the vaccinia virus gene encoding an 18-kilodalton subunit of RNA polymerase and demonstration of a 5' poly(A) leader on its early transcript.

The DNA-dependent RNA polymerase of vaccinia virus contains 8 to 10 virus-encoded polypeptides. We have mapped the gene encoding an 18-kilodalton RNA polymerase subunit to D7R, the seventh open reading frame of the HindIII D genomic subfragment. Localization of this gene was achieved by using antibody to the purified RNA polymerase for immunoprecipitation of the in vitro translation products of in vivo-synthesized early mRNA selected by hybridization to cloned DNA fragments. The identification was confirmed by translation of D7R transcripts made in vitro with bacteriophage T7 RNA polymerase. The phenotypes of two previously isolated conditionally lethal temperature-sensitive mutants that map to D7R (J. Seto, L. M. Celenza, R. C. Condit, and E. G. Niles, Virology 160:110-119, 1987) are consistent with an essential role of this subunit in late transcription. This polymerase gene, designated rpo18, predicts a polypeptide of 161 amino acids with a molecular mass of 17,892. The rpo18 gene is transcribed early in infection, even though the 5'-TAAATG-3' motif, which is conserved among many genes of the late class, is present near the RNA start site. Characterization of the 5' end of the early transcript by several different methods, including cDNA cloning, revealed a poly(A) leader with up to 14 adenylate residues, whereas only 3 are present in the corresponding location of the DNA template. Similar but somewhat longer poly(A) leaders have previously been observed in mRNAs of late genes. We noted a TAAATG motif near the initiation site of several other early genes, including the viral DNA polymerase, and carried out additional experiments to demonstrate that their early transcripts also have 5' poly(A) leaders. Thus, formation of the poly(A) leader is not exclusively a late function but apparently depends on sequences around the transcription initiation site.

Biological evaluation of glycoproteins mapping to two distinct mRNAs within the BamHI fragment 7 of pseudorabies virus: expression of the coding regions by vaccinia virus.

Several glycoproteins from the unique short region of pseudorabies have been identified and characterized. The genes encoding at least four glycoproteins (gp50, gp63, gl, and gX) are located within the BamHI fragment 7 of pseudorabies. S1 nuclease mapping was used to determine that a 2.4-kb mRNA encompasses the coding region for gp50 and gp63 and probably represents a colinear transcript for these proteins. Using the same technique, a 2.8-kb mRNA was found to encode gl. No other mRNAs were found to be encoded on the opposite strand of DNA in this region. Various recombinant vaccinia vectors were made incorporating the coding regions for these two mRNAs. Pseudorabies recombinant vaccinia infected ST cells expressed glycoproteins that co-migrated with the authentic PRV glycoproteins upon polyacrylamide electrophoresis. Intracranial or intraperitoneal inoculation of mice with the recombinant viruses constructed to contain the mRNA coding regions resulted in various degrees of protection from a lethal challenge of pseudorabies virus.

Nucleotide sequence and genome organization of canine parvovirus.

The genome of a canine parvovirus isolate strain (CPV-N) was cloned, and the DNA sequence was determined. The entire genome, including ends, was 5,323 nucleotides in length. The terminal repeat at the 3' end of the genome shared similar structural characteristics but limited homology with the rodent parvoviruses. The 5' terminal repeat was not detected in any of the clones. Instead, a region of DNA starting near the capsid gene stop codon and extending 248 base pairs into the coding region had been duplicated and inserted 75 base pairs downstream from the poly(A) addition site. Consensus sequences for the 5' donor and 3' acceptor sites as well as promotors and poly(A) addition sites were identified and compared with the available information on related parvoviruses. The genomic organization of CPV-N is similar to that of feline parvovirus (FPV) in that there are two major open reading frames (668 and 722 amino acids) in the plus strand (mRNA polarity). Both coding domains are in the same frame, and no significant open reading frames were apparent in any of the other frames of both minus and plus DNA strands. The nucleotide and amino acid homologies of the capsid genes between CPV-N and FPV were 98 and 99%, respectively. In contrast, the nucleotide and amino acid homologies of the capsid genes for CPV-N and CPV-b (S. Rhode III, J. Virol. 54:630-633, 1985) were 95 and 98%, respectively. These results indicate that very few nucleotide or amino acid changes differentiate the antigenic and host range specificity of FPV and CPV.

DNA-dependent RNA polymerase subunits encoded within the vaccinia virus genome.

Antiserum to a multisubunit DNA-dependent RNA polymerase from vaccinia virions was prepared to carry out genetic studies. This antiserum selectively inhibited the activity of the viral polymerase but had no effect on calf thymus RNA polymerase II. The specificity of the antiserum was further demonstrated by immunoprecipitation of RNA polymerase subunits from dissociated virus particles. The presence in vaccinia virus-infected cells of mRNA that encodes the polymerase subunits was determined by in vitro translation. Immunoprecipitable polypeptides with Mrs of about 135,000, 128,000, 36,000, 34,000, 31,000, 23,000, 21,000, 20,000, and 17,000 were made when early mRNA was added to reticulocyte extracts. The subunits were encoded within the vaccinia virus genome, as demonstrated by translation of early mRNA that hybridized to vaccinia virus DNA. The locations of the subunit genes were determined initially by hybridization of RNA to a series of overlapping 40-kilobase-pair DNA fragments that were cloned in a cosmid vector. Further mapping was achieved with cloned HindIII restriction fragments. Results of these studies indicated that RNA polymerase subunit genes are transcribed early in infection and are distributed within the highly conserved central portion of the poxvirus genome in HindIII fragments E, J, H, D, and A.

Homology between DNA polymerases of poxviruses, herpesviruses, and adenoviruses: nucleotide sequence of the vaccinia virus DNA polymerase gene.

A 5400-base-pair segment of the vaccinia virus genome was sequenced and an open reading frame of 938 codons was found precisely where the DNA polymerase had been mapped by transfer of a phosphonoacetate-resistance marker. A single nucleotide substitution changing glycine at position 347 to aspartic acid accounts for the drug resistance of the mutant vaccinia virus. The 5' end of the DNA polymerase mRNA was located 80 base pairs before the methionine codon initiating the open reading frame. Correspondence between the predicted Mr 108,577 polypeptide and the 110,000 purified enzyme indicates that little or no proteolytic processing occurs. Extensive homology, extending over 435 amino acids, was found upon comparing the DNA polymerase of vaccinia virus and DNA polymerase of Epstein-Barr virus. A highly conserved sequence of 14 amino acids in the carboxyl-terminal regions of the above DNA polymerases is also present at a similar location in adenovirus DNA polymerase. This structure, which is predicted to form a turn flanked by beta-pleated sheets, may form part of an essential binding or catalytic site that accounts for its presence in DNA polymerases of poxviruses, herpesviruses, and adenoviruses.

Transcriptional mapping of the vaccinia virus DNA polymerase gene.

Transcriptional analysis of the vaccinia virus DNA polymerase gene revealed the presence of overlapping RNAs. Cloned DNA fragments, previously shown to lie within the DNA polymerase gene (E. V. Jones and B. Moss, J. Virol. 49:72-77, 1984) hybridized to early RNA species of ca. 3,400 and 3,700 nucleotides. Nuclease S1 analysis was used to determine the direction of transcription and more precisely map the mRNAs. A single 5' end and two major and one minor 3' ends were detected.

Mapping of the vaccinia virus DNA polymerase gene by marker rescue and cell-free translation of selected RNA.

The previous demonstration that a phosphonoacetate (PAA)-resistant (PAAr) vaccinia virus mutant synthesized an altered DNA polymerase provided the key to mapping this gene. Marker rescue was performed in cells infected with wild-type PAA-sensitive (PAAs) vaccinia by transfecting with calcium phosphate-precipitated DNA from a PAAr mutant virus. Formation of PAAr recombinants was measured by plaque assay in the presence of PAA. Of the 12 HindIII fragments cloned in plasmid or cosmid vectors, only fragment E conferred the PAAr phenotype. Successive subcloning of the 15-kilobase HindIII fragment E localized the marker within a 7.5-kilobase BamHI-HindIII fragment and then within a 2.9-kilobase EcoRI fragment. When the latter was digested with ClaI, marker rescue was not detected, suggesting that the PAAr mutation mapped near a ClaI site. The sensitive ClaI site was identified by cloning partial ClaI-EcoRI fragments and testing them in the marker rescue assay. The location of the DNA polymerase gene, about 57 kilobases from the left end of the genome, was confirmed by cell-free translation of mRNA selected by hybridization to plasmids containing regions of PAAr vaccinia DNA active in marker rescue. A 100,000-dalton polypeptide that comigrated with authentic DNA polymerase was synthesized. Correspondence of the in vitro translation product with purified vaccinia DNA polymerase was established by peptide mapping.

Persistent infection of L cells with vesicular stomatitis virus: evolution of virus populations.

A previous report (Youngner et al., J. Virol. 19:90-101, 1976) documented that noncytocidal persistent infection can be established with wild-type vesicular stomatitis virus (VSV) in mouse L cells at 37 degrees C and that a rapid selection of RNA(-), group I temperature-sensitive (ts) mutants consistently occurs in this system. To assess the selective advantage of the RNA(-)ts phenotype, evolution of the virus population was studied in persistent infections initiated in L cells by use of VSV ts 0 23 and ts 0 45, RNA(+) mutants belonging to complementation groups III and V. In L cells persistently infected with ts 0 23, the ts RNA(+) virus population was replaced gradually by viruses which had a ts RNA(-) phenotype. VSV ts 0 45 (V) has another marker in addition to reduced virus yield at 39.5 degrees C: a defective protein (G) which renders virion infectivity heat labile at 50 degrees C. Persistent infections initiated with this virus (ts, heat labile, RNA(+)) evolved into a virus population which was ts, heat resistant, and RNA(-). These findings suggest that the ts phenotype itself is not sufficient to stabilize the VSV population in persistently infected L cells and also indicate that the ts RNA(-) phenotype may have a unique selective advantage in this system. In addition to the selection of ts RNA(-) mutants, other mechanisms which also might operate in the maintenance of persistent VSV infections of L cells were explored. Whereas defective-interfering particles did not seem to mediate the carrier state, evidence was obtained that interferon may play a role in the regulation of persistent infections of L cells with VSV.

Post-halothane jaundice in relation to previous administration of halothane.

The time interval since previous anaesthesia was compared in a surgical population in South Wales and in patients who developed jaundice after halothane. There was a significant difference in the pattern of time interval since previous general anaesthetics in the surgical population and in those patients who developed jaundice after halothane. In the group who developed jaundice there was an "excess" of patients who had had a previous halothane anaesthetic within four weeks. Halothane should if possible be avoided in patients who have had it before, particularly if this was within the previous four weeks. In the case of repeat halothane anaesthetics within four weeks, the risk seems to lie between 1 in 6,000 and 1 in 22,000.