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Biomedical subjects

John M Taylor

Publications and source records attributed to John M Taylor.

At least 19 recordsLinked to original sources
PubMed2006

The vaccinia virus protein F1L interacts with Bim and inhibits activation of the pro-apoptotic protein Bax.

Vaccinia virus, the prototypic member of the orthopoxvirus genus, encodes the mitochondrial-localized protein F1L that functions to protect cells from apoptotic death and inhibits cytochrome c release. We previously showed that F1L interacts with the pro-apoptotic Bcl-2 family member Bak and inhibits activation of Bak following an apoptotic stimulus (Wasilenko, S. T., Banadyga, L., Bond, D., and Barry, M. (2005) J. Virol. 79, 14031-14043). In addition to Bak, the pro-apoptotic protein Bax is also capable of initiating cytochrome c release suggesting that vaccinia virus infection could also inhibit Bax activity. Here we show that F1L inhibits the activity of the pro-apoptotic protein Bax by inhibiting oligomerization and N-terminal activation of Bax. F1L expression also inhibited the subcellular redistribution of Bax to the mitochondria and the insertion of Bax into the outer mitochondrial membrane. The ability of F1L to inhibit Bax activation does not require Bak, because F1L expression inhibited cytochrome c release and Bax activation in Bak-deficient cells. No interaction between Bax and F1L was detected during infection, suggesting that F1L functions upstream of Bax activation. Notably, F1L was capable of interacting with the BH3-only protein BimL as shown by co-immunoprecipitation, and F1L expression inhibited apoptosis induced by BimL. These studies suggest that, in addition to interacting with the pro-apoptotic protein Bak, F1L also functions to indirectly inhibit the activation of Bax, likely by interfering with the pro-apoptotic activity of BH3-only proteins such as BimL.

Animals↗
PubMed2006

Restoration in vivo of defective hepatitis delta virus RNA genomes.

The 1679-nt single-stranded RNA genome of hepatitis delta virus (HDV) is circular in conformation. It is able to fold into an unbranched rodlike structure via intramolecular base-pairing. This RNA is replicated by host RNA polymerase II (Pol II). Such transcription is unique, because Pol II is known only for its ability to act on DNA templates. The present study addressed the ability of the HDV RNA replication to tolerate insertions of up to 1000 nt of non-HDV sequence either at an end of the rodlike RNA structure or at a site embedded within the rod. The insertions did not interfere with the ability of primary transcripts to be processed in vivo by ribozyme cleavage and ligation. The insertions greatly reduced the ability of genomes to replicate. However, when total RNA from such transfected cells was used to transfect new recipient cells, replicating HDV RNAs could be detected by Northern analyses. The size of the emerged RNAs was consistent with loss of the inserted sequences. RT-PCR, cloning, and sequencing showed that recovery involved removal of inserted sequences with or without small deletions of adjacent RNA sequences. Such restoration of the RNA genome is consistent with a model requiring intramolecular template-switching (RNA recombination) during RNA-directed transcription, in combination with biological selection for maintenance of the rodlike structure of the template.

Base Pairing↗
PubMed2006

Near death experiences: poxvirus regulation of apoptotic death.

Apoptosis, or programmed cell death, plays a critical role in the elimination of virus-infected cells. As a result, a growing number of viruses encode numerous potent anti-apoptotic proteins to counteract apoptosis in an effort to prolong their own survival. This review describes the numerous mechanisms by which poxviruses inhibit apoptosis thereby modulating life and death of the cell.

Animals↗
PubMed2006

Hepatitis delta virus.

Hepatitis delta virus (HDV) is a sub-viral agent that is dependent for its life cycle on hepatitis B virus (HBV). The help it obtains from HBV is limited to the sharing of envelope proteins. These proteins are needed to facilitate the assembly of the HDV genome into new virus particles, and in turn, to allow the attachment and entry of HDV into new host cells. In other respects, the replication of the small single-stranded circular RNA genome of HDV is independent of HBV. HDV genome replication produces two forms of a RNA-binding protein known as the long and small delta antigens (Ag). All other proteins needed for HDV genome replication, especially the RNA-directed RNA polymerase activity, are provided by the host cell. This mini-review article is a mixture of personal perspective and speculations about the future of HDV research. It starts with a brief overview of HDV and its replication, notes some of the major unresolved questions, and directs the interested reader to more detailed reviews.

Animals↗
PubMed2006

Critical care challenges in orthopedic surgery patients.

OBJECTIVES: To review common and life-threatening complications resulting in intensive care unit admission for patients undergoing orthopedic surgery procedures. To identify specific diagnostic techniques and treatment modalities that may improve the outcome of critically ill orthopedic surgery patients. DESIGN: A review of the current literature regarding the care of orthopedic surgery patients was performed. RESULTS: Orthopedic surgery patients are vulnerable to a number of complications, particularly pulmonary complications related to scoliosis surgery, embolic complications of joint arthroplasty, and complications related to the use of opioids and sedatives in an elderly population. They also are susceptible to transfusion-related complications such as transfusion-related acute lung injury and transfusion-associated circulatory overload. Specific strategies for management of these complex patients are identified. CONCLUSIONS: Orthopedic surgery patients represent a significant and growing proportion of patients in intensive care units. They develop unique complications, and management of these complications requires understanding of preoperative co-morbidities, intraoperative management, and early recognition and treatment of postoperative complications. Prevention and early identification of these complications are the most efficacious routes to improving outcomes in this patient population.

Airway Obstruction↗
PubMed2005

High-level lipoprotein [a] expression in transgenic mice: evidence for oxidized phospholipids in lipoprotein [a] but not in low density lipoproteins.

Efforts to elucidate the role of lipoprotein [a] (Lp[a]) in atherogenesis have been hampered by the lack of an animal model with high plasma Lp[a] levels. We produced two lines of transgenic mice expressing apolipoprotein [a] (apo[a]) in the liver and crossed them with mice expressing human apolipoprotein B-100 (apoB-100), generating two lines of Lp[a] mice. One had Lp[a] levels of approximately 700 mg/dl, well above the 30 mg/dl threshold associated with increased risk of atherosclerosis in humans; the other had levels of approximately 35 mg/dl. Most of the LDL in mice with high-level apo[a] expression was covalently bound to apo[a], but most of the LDL in the low-expressing line was free. Using an enzyme-linked sandwich assay with monoclonal antibody EO6, we found high levels of oxidized phospholipids in Lp[a] from high-expressing mice but not in LDL from low-expressing mice or in LDL from human apoB-100 transgenic mice (P <0.00001), even though all mice had similar plasma levels of human apoB-100. The increase in oxidized lipids specific to Lp[a] in high-level apo[a]-expressing mice suggests a mechanism by which increased circulating levels of Lp[a] could contribute to atherogenesis.

Animals↗
PubMed2005

Development of a novel system to study hepatitis delta virus genome replication.

Hepatitis delta virus (HDV) genome replication requires the virus-encoded small delta protein (deltaAg). During replication, nucleotide sequence changes accumulate on the HDV RNA, leading to the translation of deltaAg species that are nonfunctional or even inhibitory. A replication system was devised where all deltaAg was conditionally provided from a separate and unchanging source. A line of human embryonic kidney cells was stably transfected with a single copy of cDNA encoding small deltaAg, with expression under tetracycline (TET) control. Next, HDV genome replication was initiated in these cells by transfection with a mutated RNA unable to express deltaAg. Thus, replication of this RNA was under control of the TET-inducible deltaAg. In the absence of TET, there was sufficient deltaAg to allow a low level of HDV replication that could be maintained for at least 1 year. When TET was added, both deltaAg and genomic RNA increased dramatically within 2 days. With clones of such cells, designated 293-HDV, the burst of HDV RNA replication interfered with cell cycling. Within 2 days, there was a fivefold enhancement of G1/G0 cells relative to both S and G2/M cells, and by 6 days, there was extensive cell detachment and death. These findings and those of other studies that are under way demonstrate the potential applications of this experimental system.

Cell Line↗
PubMed2005

Evolution of hepatitis delta virus RNA genome following long-term replication in cell culture.

Previous studies have defined a novel cell culture system in which a modified RNA genome of hepatitis delta virus (HDV) is able to maintain a low level of continuous replication for at least 1 year, using a separate and limited DNA-directed source of mRNA for the essential small delta protein. This mode of replication is analogous to that used by plant viroids. An examination was made of the nucleotide changes that accumulated on the HDV RNA during 1 year of replication. The length of the RNA genome was maintained, except for some single-nucleotide deletions and insertions. There was an abundance of single-nucleotide substitutions, with a 22-fold excess of these being base transitions rather than transversions. Of the detected transitions, at least 70% were consistent with being the consequences of posttranscriptional RNA editing by an adenosine deaminase acting on RNA. The remainder of the changes, including the single-nucleotide insertions and deletions, are likely to be the consequence of misincorporation during transcription. In addition, an intermolecular competition assay was used to show that the majority of the genomes present after 1 year of replication were essentially as competent in replication as the original single HDV RNA sequence that was used to initiate the genome replication. A model is provided to explain how, in this experimental system, the observed single-nucleotide changes were essentially neutral in terms of their effect on the ability of the HDV genome to carry out continued rounds of replication.

Base Sequence↗
PubMed2004

Reconstitution in cultured cells of replicating HDV RNA from pairs of less than full-length RNAs.

The genome of hepatitis delta virus (HDV) is a small single-stranded circular RNA that is replicated via RNA-directed RNA synthesis. This makes use of a host RNA polymerase, probably pol II, that normally transcribes DNA templates. In vivo, the host polymerase can initiate replication from transfected linear RNAs using intramolecular template-switching. The present studies report that the polymerase could also achieve intermolecular switching leading to "reconstitution" of full-length HDV RNAs following transfection with two linear RNAs that were less than full length and yet lacking different regions of the genome. These two RNAs were synthesized in vitro, gel purified, pre-annealed, and then transfected into delta293, a cell line conditionally expressing the small delta antigen that is essential for HDV replication. Northern analyses of total RNA harvested from transfected cells detected the accumulation of full-length HDV genomic and antigenomic RNAs. Such reconstitution of full-length replicating HDV RNA was also achieved using nine other pairs of antigenomic RNAs and three pairs of genomic RNAs. Annealing of the RNAs prior to transfection was required for detectable HDV reconstitution. A second cell line, Huh7, also supported reconstitution when a pair of RNAs was cotransfected together with mRNA for the small delta protein. Taken together, these results support a model that observed genome reconstitution is a special form of recombination involving intermolecular template switches and they provide insights into the mechanism of RNA-directed RNA transcription catalyzed by a host RNA polymerase.

Cell Line↗
PubMed2004

miR-122, a mammalian liver-specific microRNA, is processed from hcr mRNA and may downregulate the high affinity cationic amino acid transporter CAT-1.

These studies show that miR-122, a 22-nucleotide microRNA, is derived from a liver-specific noncoding polyadenylated RNA transcribed from the gene hcr. The exact sequence of miR-122 as well as the adjacent secondary structure within the hcr mRNA are conserved from mammalian species back to fish. Levels of miR-122 in the mouse liver increase to half maximal values around day 17 of embryogenesis, and reach near maximal levels of 50,000 copies per average cell before birth. Lewis et al. (2003) predicted the cationic amino acid transporter (CAT-1 or SLC7A1) as a miR-122 target. CAT-1 protein and its mRNA are expressed in all mammalian tissues but with lower levels in adult liver. Furthermore, during mouse liver development CAT-1 mRNA decreases in an almost inverse correlation with miR-122. Eight potential miR-122 target sites were predicted within the human CAT-1 mRNA, with six in the 3'-untranslated region. Using a reporter construct it was found that just three of the predicted sites, linked in a 400-nucleotide sequence from human CAT-1, acted with synergy and were sufficient to strongly inhibit protein synthesis and reduce mRNA levels. In summary, these studies followed the accumulation during development of miR-122 from its mRNA precursor, hcr, through to identification of what may be a specific mRNA target, CAT-1.

Animals↗
PubMed2004

Features affecting the ability of hepatitis delta virus RNAs to initiate RNA-directed RNA synthesis.

In models of the replication of human hepatitis delta virus (HDV) RNA, it is generally assumed that circular RNAs are the only templates. However, noncircular HDV RNAs are also produced during replication, and it is known that replication can be initiated by transfection with noncircular RNAs. Therefore, strategies were devised to determine the relative ability of different HDV RNA species to initiate RNA replication. One strategy used in vivo intermolecular competition following cotransfection into cells, between two sequence-marked HDV RNA species. Circular RNA templates were found to be at least severalfold more efficient than a dimeric linear template. Unit-length linear species, that is, equivalent to circles opened at different sites, were in most cases but not always of efficiency comparable to that of each other. Greater-than-unit-length linear species were more efficient than unit-length species, presumably because of the increased opportunities for template switching. Genomic linear RNAs were generally of initiation ability comparable to that of antigenomic RNAs. A second strategy measured the ability of initiation to occur on different regions of HDV RNAs that were twice the unit length. In summary, results from these two experimental strategies make clear that linear HDV RNA species, as well as circles, can contribute to the overall process of HDV genome replication. In addition, the results from the two experimental strategies provided information on the impact of template switching during RNA-directed transcription.

Cell Line, Tumor↗
PubMed2004

Construction of a herpes simplex virus type 1 mutant with only a three-nucleotide change in the branchpoint region of the latency-associated transcript (LAT) and the stability of its two-kilobase LAT intron.

Previous studies using a eukaryotic expression system indicated that the unusual stability of the latency-associated transcript (LAT) intron was due to its nonconsensus branchpoint sequence (T.-T Wu, Y.-H. Su, T. M. Block, and J. M. Taylor, Virology, 243:140-149, 1998). The present study investigated the role of the branchpoint sequence in the stability of the intron expressed from the herpes simplex virus type 1 (HSV-1) genome and the role of LAT intron stability in the HSV-1 life cycle. A branchpoint mutant called Sy2 and the corresponding rescued viruses, SyRA and SyRB, were constructed. To preserve the coding sequence of the immediate early gene icp0, which overlaps with the branchpoint region of the 2-kb LAT, a 3-nucleotide mutation into the branchpoint region of the 2-kb LAT was introduced, resulting in a branchpoint that is 85% identical to the consensus intron branchpoint sequence of eukaryotic cells. As anticipated, there was a 90- to 96-fold reduction in 2-kb LAT accumulation following productive infection in tissue culture and latent infection in mice with Sy2, as determined by Northern blot analysis. These results clearly suggest that the accumulation of the 2-kb intron in tissue culture and in vivo is, at least in part, due to the nonconsensus branchpoint sequence of the LAT intron. Interestingly, a failure to accumulate LAT was associated with greater progeny production of Sy2 at a low multiplicity of infection (0.01) in tissue culture, but not in mice. However, the ability of mutant Sy2 to reactivate from trigeminal ganglia (TG) derived from latently infected mice was indistinguishable from that of wild-type virus, as assayed in the mouse TG explant reactivation system.

Animals↗
PubMed2004

Alternative processing of hepatitis delta virus antigenomic RNA transcripts.

Intrinsic to the life cycle of hepatitis delta virus (HDV) is the fact that its RNAs undergo different forms of posttranscriptional RNA processing. Transcripts of both the genomic RNA and its exact complement, the antigenomic RNA, undergo ribozyme cleavage and RNA ligation. In addition, antigenomic RNA transcripts can undergo 5' capping, 3' polyadenylation, and even RNA editing by an adenosine deaminase. This study focused on the processing of antigenomic RNA transcripts. Two approaches were used to study the relationship between the events of polyadenylation, ribozyme cleavage, and RNA ligation. The first represented an examination under more controlled conditions of mutations in the poly(A) signal, AAUAAA, which is essential for this processing. We found that when a separate stable source of deltaAg-S, the small delta protein, was provided, the replication ability of the mutated RNA was restored. The second approach involved an examination of the processing in transfected cells of specific Pol II DNA-directed transcripts of HDV antigenomic sequences. The DNA constructs used were such that the RNA transcripts were antigenomic and began at the same 5' site as the mRNA produced during RNA-directed HDV genome replication. A series of such constructs was assembled in order to test the relative abilities of the transcripts to undergo processing by polyadenylation or ribozyme cleavage at sites further 3' on a multimer of HDV sequences. The findings from the two experimental approaches led to significant modifications in the rolling-circle model of HDV genome replication.

Cell Line↗
PubMed2003

Effects on lipoprotein subclasses of combined expression of human hepatic lipase and human apoB in transgenic rabbits.

OBJECTIVE: The effects of combined expression of human hepatic lipase (HL) and human apolipoprotein B (apoB) on low-density lipoprotein (LDL) subclasses were examined in rabbits, a species naturally deficient in HL activity. METHODS AND RESULTS: In apoB-transgenic rabbit plasma, >80% of the protein was found in the 1.006- to 1.050-g/mL fraction. Gradient gel electrophoresis (GGE) of this fraction revealed two distinct species, designated large and small LDL. A denser fraction (d=1.050 to 1.063 g/mL) contained small LDL as well as another discrete LDL subspecies, designated very small LDL. Expression of HL resulted in reductions in protein concentrations in the 1.006- to 1.050-g/mL density-gradient subfractions containing large (6.5+/-4.1 versus 32.6+/-12.0 mg/dL, P<0.005) and small LDL (59.6+/-17.4 versus 204.3+/-50.3 mg/dL, P<0.002). A concomitant small but not significant increase in protein concentration in the denser LDL fraction (48.0+/-28.2 versus 44.6+/-18.2 mg/dL) was due primarily to an increase in very small LDL (25.9+/-3.1 versus 9.6+/-5.4% of total LDL GGE densitometric area, P<0.002). CONCLUSIONS: These findings support a direct role for HL in regulating total plasma LDL concentrations as well as in the production of smaller, denser LDL from larger, more buoyant precursors.

Animal Feed↗
PubMed2003

Therapy for HDV!

Explore the source record for details and available documents.

Animals↗
PubMed2003

Replication of human hepatitis delta virus: recent developments.

In a natural setting, hepatitis delta virus (HDV) is only found in patients that are also infected with hepatitis B virus (HBV). In hepatocytes infected with these two viruses, HDV RNA genomes are assembled using the envelope proteins of HBV. Since 1986, we have known that HDV has a small single-stranded RNA genome with a unique circular conformation that is replicated using a host RNA polymerase. These and other features make HDV and its replication unique, at least among agents that infect animals. This mini-review focuses on advances gained over the last 2-3 years, together with an evaluation of HDV questions that are either unsolved or not yet solved satisfactorily.

Gene Silencing↗