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Biomedical subjects

Richard Adair

Publications and source records attributed to Richard Adair.

11 recordsLinked to original sources
PubMed2006

Human cytomegalovirus temporally regulated gene expression in differentiated, immortalized retinal pigment epithelial cells.

BACKGROUND: Human cytomegalovirus (HCMV) replication in epithelial cells is crucial for its pathogenesis. To date, HCMV gene expression has been primarily studied in human foreskin fibroblasts (HFFs), although their importance for HCMV pathogenesis remains unclear. Primary retinal pigment epithelial (RPE) cells are permissive for HCMV. OBJECTIVES: Our objectives were to determine the production of alternatively processed HCMV major immediate-early (MIE) and UL37 RNAs and their essential products in infected, terminally differentiated immortalized RPE (hTERT-RPE) cells. STUDY DESIGN: hTERT-RPE cells were studied because of their notable similarities with primary RPE cells, and because they overcome key limitations of primary cells. hTERT-RPE cells were terminally differentiated in vitro and infected with HCMV. Total RNA or cell proteins were analyzed at various times post-infection. RESULTS: We show for the first time that HCMV-infected, differentiated hTERT-RPE cells produce IE1, IE2, UL37 exon 1 (UL37x1) and UL37 alternatively spliced RNAs, albeit with abundances and kinetics distinct from those observed in HCMV-infected HFFs. IE1-72 was produced in HCMV-infected, differentiated hTERT-RPEs within 24h post-infection (hpi); whereas, IE2-86 and pUL37x1 were produced within 72 hpi. IE2-86 was detected after IE1-72 even though its transcript appeared first. Early/late (pp65) and late (pp28) proteins were produced within 96-120 hpi. CONCLUSIONS: The temporal cascade of HCMV gene expression was observed in infected, differentiated hTERT-RPE cells. Moreover, HCMV IE RNAs are alternatively and accurately processed in differentiated hTERT-RPE cells. However, the delayed temporal expression suggests further regulation of HCMV gene expression at post-transcriptional/translational levels in differentiated hTERT-RPE cells.

Alternative Splicing↗
PubMed2006

Acetylated Tat regulates human immunodeficiency virus type 1 splicing through its interaction with the splicing regulator p32.

The human immunodeficiency virus type 1 (HIV-1) potent transactivator Tat protein mediates pleiotropic effects on various cell functions. Posttranslational modification of Tat affects its activity during viral transcription. Tat binds to TAR and subsequently becomes acetylated on lysine residues by histone acetyltransferases. Novel protein-protein interaction domains on acetylated Tat are then established, which are necessary for both sustained transcriptional activation of the HIV-1 promoter and viral transcription elongation. In this study, we investigated the identity of proteins that preferentially bound acetylated Tat. Using a proteomic approach, we identified a number of proteins that preferentially bound AcTat, among which p32, a cofactor of splicing factor ASF/SF-2, was identified. We found that p32 was recruited to the HIV-1 genome, suggesting a mechanism by which acetylation of Tat may inhibit HIV-1 splicing needed for the production of full-length transcripts. Using Tat from different clades, harboring a different number of acetylation sites, as well as Tat mutated at lysine residues, we demonstrated that Tat acetylation affected splicing in vivo. Finally, using confocal microscopy, we found that p32 and Tat colocalize in vivo in HIV-1-infected cells.

Acetylation↗
PubMed2006

Access to health care: differences between insured and uninsured patients in south Minneapolis.

This article reports the results of a survey of 575 patients who were seen at 2 clinics in south Minneapolis. About half had health insurance. We found a strong connection between not having insurance and going without medical care. We also found that when the uninsured do go for care, they often fail to take prescribed medications because of the cost. Our findings also contradicted some prevalent thinking about the uninsured: that they are unemployed, that they refuse employer-sponsored insurance, and that they can easily get care at free clinics.

Adult↗
PubMed2005

Posting guidelines: a practical and effective way to promote appropriate hypertension treatment.

BACKGROUND: Despite publication and periodic updating of treatment guidelines, hypertension remains undertreated in the United States, and physicians underuse recommended drugs. METHODS: Hypertension treatment guidelines were summarized and posted in five places in a hospital-based primary care clinic staffed by internists and internal medicine residents. Costs and recommended doses of five commonly used antihypertensive drugs were included. The charts of all 253 patients seen during a four-month period with a diagnosis of hypertension were analyzed. Blood pressures and physician prescribing habits were compared at baseline and at 8, 12, and 16 months after posting the guidelines. RESULTS: The number of patients with blood pressures < 140/90 mm Hg increased from 41% to 58%, p = .001. Median (IQR) systolic pressure fell from 143 (119-167) to 137 (116-158) mm Hg, p < .0001 and diastolic pressure from 78 (65-91) to 77 (64-90) mm Hg, p = .0002. Physicians prescribed more recommended drugs, more total antihypertensive drugs, larger doses of hydrochlorothiazide and lisinopril, and more inexpensive drugs. The total cost of antihypertensive drugs per patient increased slightly. CONCLUSION: Regular exposure to clinical guidelines, presented in a practical and simple way, can change physician behavior and improve patient care.

Adult↗
PubMed2004

Alteration of cellular RNA splicing and polyadenylation machineries during productive human cytomegalovirus infection.

Alternative processing of human cytomegalovirus (HCMV) UL37 pre-mRNA predominantly produces the unspliced UL37 exon 1 (UL37x1) RNA and multiple, lower abundance, alternatively spliced UL37 RNAs. The relative abundance of UL37x1 unspliced RNA is surprising because it requires the favoured use of a polyadenylation signal within UL37 intron 1, just upstream of the UL37 exon 2 (UL37x2) acceptor. Here, it was shown that a downstream element (DSE) in UL37x2 strongly enhanced processing at the UL37x1 polyadenylation site, but did not influence UL37x1-x2 splicing. There was a potential binding site (UCUU) for polypyrimidine tract-binding protein (PTB) at the UL37x1 polyadenylation/cleavage site and its mutation to UGGG reduced both polyadenylation and splicing of UL37x1-x2 minigene pre-mRNA, suggesting a role in both RNA processing events. To determine whether lytic HCMV infection altered the balance of RNA processing factors, which bind to UL37 pre-mRNA cis elements, these were investigated in permissively infected primary and immortalized human diploid fibroblasts (HFFs) and epithelial cells. Induction of polyadenylation factors in HCMV-infected, serum-starved (G(0)) HFFs was also investigated. Permissive HCMV infection consistently increased, albeit with different kinetics, the abundance of cleavage stimulation factor 64 (CstF-64) and PTB, and altered hypo-phosphorylated SF2 in different cell types. Moreover, the preponderance of UL37x1 RNA increased during infection and correlated with CstF-64 induction, whereas the complexity of the lower abundance UL37 spliced RNAs transiently increased following reduction of hypo-phosphorylated SF2. Collectively, multiple UL37 RNA polyadenylation cis elements and induced cellular factors in HCMV-infected cells strongly favoured the production of UL37x1 unspliced RNA.

Binding Sites↗
PubMed2003

Human cytomegalovirus UL37 immediate early target minigene RNAs are accurately spliced and polyadenylated.

The human cytomegalovirus (HCMV) UL36-38 immediate early (IE) locus encodes proteins required for virus growth. The UL37 IE promoter drives production of differentially spliced and unspliced RNAs. To study their post-transcriptional processing, we generated target minigenes encoding each UL37 RNA splicing substrate. Target 1 RNA, spanning UL37 exon 1 (x1) donor and 2 (x2) acceptor as well as adjacent intronic sequences, but not the UL38 gene, accurately reproduced UL37 x1/x2 RNA splicing in transfected permissive cells. Surprisingly, deletion of distal intronic sequences nt -82 to -143 from the UL37x2 acceptor resulted in aberrant splicing to an upstream non-consensus exonic donor. Target 1 RNAs carry the UL37x1 polyadenylation (PA) signal and site as well as a downstream SV40 early PA signal. Both the UL37x1 and SV40 PA signals are used in wild-type target 1 RNAs but inhibited in UL37x1 PA signal mutants. Alternative RNA splicing of UL37 exons 2 to 3 or 3A as well as exons 3 to 4, observed in HCMV mature UL37 and UL36 spliced RNAs, is accurately reproduced with target minigene RNAs carrying the corresponding UL37 exonic and intronic sequences. Moreover, alternative splicing using two novel UL37 exon 3 consensus splice donors (di and dii) was found in target and in HCMV-infected cell RNA. These results demonstrate that: (i) target minigene RNAs accurately recapitulate the processing of UL37 IE RNAs in the HCMV-infected cell; (ii) precise UL37x1 donor selection is modulated by 3'-distal UL37 intronic sequences; and (iii) UL37 exon 3 contains multiple alternative consensus splice donors.

Base Sequence↗
PubMed2003

Complex alternative processing of human cytomegalovirus UL37 pre-mRNA.

Differentially processed human cytomegalovirus (HCMV) UL37 RNAs encode biologically significant proteins. Due to the recent discovery of alternative UL37 exon 3 (UL37x3) splice donors, permissively infected cells were thoroughly examined for additional alternatively spliced UL37 RNAs. Newly described donors within UL37 exon 1 (nt 52520) and intron 1 (nt 52209) as well as UL37x3 di (nt 50770) and dii (nt 50782) were differentially spliced to known downstream UL37 acceptors. The alternatively spliced UL37(S), UL37(L), UL37(di) and UL37d(ii) RNAs predictably encode proteins of 83, 163, 217 and 213 residues, respectively, which share UL37x1 N-terminal sequences but differ downstream in their C termini. Moreover, temporal expression of the alternatively spliced UL37 RNAs differs during HCMV infection. The complexity of UL37 pre-mRNA processing is evidenced by the detection of 11 UL37 spliced and unspliced UL37x1 RNAs in HCMV-infected cells. Based upon these data, a revised HCMV UL37 gene map is presented, which incorporates all RNA species detected during permissive infection.

Alternative Splicing↗
PubMed2003

Convergence of RNA cis elements and cellular polyadenylation factors in the regulation of human cytomegalovirus UL37 exon 1 unspliced RNA production.

The human cytomegalovirus (HCMV) UL36-38 immediate early (IE) locus encodes proteins required for its growth. The UL37 promoter drives production of an unspliced and several alternatively spliced RNAs. The UL37 exon 1 (UL37x1) unspliced RNA is abundant from IE to late times of HCMV infection, whereas the UL37 spliced RNAs are markedly less abundant. Production of the UL37x1 unspliced RNA requires polyadenylation (PA) at nucleotide 50998, which lies within intron 1, upstream of the UL37 exon 2 (UL37x2) acceptor. The physical proximity of its cis elements suggests steric hindrance between PA and splicing machineries for UL37 pre-mRNA. To test this possibility, we generated site-specific mutants in Target 1 PA and RNA splicing cis elements and compared the PA and splicing efficiencies of mutant RNAs with those of wild-type RNA. The mutually exclusive processing events of UL37x1 PA and UL37x1-UL37x2 splicing have been accurately recapitulated in transfected permissive human fibroblasts (HFFs) expressing a Target 1 minigene RNA, which contains the required splicing and PA cis elements. Two mutants in the invariant PA signal dramatically decreased UL37x1 PA as expected and, concomitantly, increased the efficiency of UL37x1-UL37x2 RNA splicing. Consistent with these results, changes to consensus UL37x1 donor and UL37x2 acceptor sites increased the efficiency of UL37x1-UL37x2 RNA splicing but decreased the efficiency of UL37x1 PA. Moreover, HCMV infection of HFFs increased the abundance of the PA cleavage stimulatory factor CstF-64, the potent splicing suppressor PTB, and the hypophosphorylated form of the splicing factor SF2 at 4 h postinfection. Induction of these factors further favors production of the UL37x1 unspliced RNA over that of the spliced RNAs. Taken together, these results suggest that there is a convergence in UL37 RNA regulation by cis elements and cellular proteins which favors production of the UL37x1 unspliced RNA during HCMV infection at the posttranscriptional level.

Alternative Splicing↗
PubMed2002

The products of human cytomegalovirus genes UL23, UL24, UL43 and US22 are tegument components.

We have investigated the human cytomegalovirus (HCMV) US22 gene family members UL23, UL24, UL43 and US22. Specific antibodies were generated to identify pUL23 (33 kDa), pUL24 (40 kDa) and pUL43 (48 kDa), while pUS22 was identified by monoclonal antibody HWLF1. A C-terminally truncated UL43 product (pUL43t; 21 kDa) produced by a deletion mutant was also investigated. The UL24 and UL43 genes were expressed with early-late (gamma1) and true-late (gamma2) kinetics, respectively. Immunoblot and immuno-EM studies demonstrated that pUL23, pUL24, pUL43 and pUS22 were virion tegument components. Immunofluorescence and immuno-EM studies showed that pUL23, pUL24, pUL43 and pUL43t were located in cytoplasmic protein aggregates, manifesting two forms: complex juxtanuclear structures and smaller, membrane-bound aggregates resembling dense bodies. The complex-type aggregate is a putative site of particle maturation. Because pUL43t was present in protein aggregates, but under-represented in virus particles compared to pUL43, it was concluded that N-terminal sequences target pUL43 to protein aggregates and that C-terminal sequences are important for incorporation into particles. Since three other US22 family products (pUL36, pTRS1 and pIRS1) are documented tegument components, at least seven of the twelve US22 family genes encode tegument proteins, suggesting that the products of the remaining five genes might be similarly located. These findings demonstrate a common biological feature among most, if not all, US22 family proteins and implicate the family in events occurring immediately after virus penetration.

Antibodies, Monoclonal↗