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Biomedical subjects

W Yu

Publications and source records attributed to W Yu.

At least 289 records · Page 16Linked to original sources
PubMed1995

Analysis of leukotrienes, lipoxins, and monooxygenated metabolites of arachidonic acid by reversed-phase high-pressure liquid chromatography.

Arachidonic acid can be converted to a large number of metabolites by various lipoxygenases, cyclooxygenase, and other enzymes. Because of the complex profiles of products formed by many types of cells, high-pressure liquid chromatography (HPLC) has proved to be an invaluable technique for their purification and analysis. In the present study we have developed improved methods for the analysis of complex mixtures of eicosanoids by HPLC using binary gradients containing trifluoroacetic acid (TFA), which allows considerable manipulation of the retention times of the cysteine-containing leukotrienes (LTs) LTC4, LTD4, and LTE4 relative to those of other eicosanoids. With a gradient between 0.003 and 0.005% TFA and a 4.6-mm-i.d. column of Spherisorb ODS-2, cysteinyl-LTs are very well resolved from one another and are separated as a group with retention times longer than those of all other major eicosanoids. These conditions can be used for the analysis of prostaglandin B2 (PGB2), LTB4, monohydroxyeicosatetraenoic acids (HETEs), and cysteinyl-LTs in only 30 min. Slightly longer analysis times must be used for the separation of more polar eicosanoids such as hydroxy metabolites of LTB4 and lipoxins. We have also developed methods for the analysis of eicosanoids using a midbore (3.2 mm i.d.) column containing Spherisorb ODS-2, which improves sensitivity and reduces solvent consumption. In this case higher concentrations (0.04 to 0.05%) of TFA have been used, resulting in retention times for cysteinyl-LTs between those of the cyclooxygenase product 12-hydroxy-5,8,10-heptadecatrienoic acid and the HETEs. This approach permits analysis of PGB2, LTB4, HETEs, and cysteinyl-LTs in only 20 min. Samples which also contain hydroxy-LTB4 and lipoxins can be analyzed in 40 min. The above techniques are highly reproducible and give baselines which are free of interfering peaks.

Animals↗
PubMed1995

Estimates of volumetric bone density from projectional measurements improve the discriminatory capability of dual X-ray absorptiometry.

To determine whether estimates of volumetric bone density from projectional scans of the lumbar spine have weaker associations with height and weight and stronger associations with prevalent vertebral fractures than standard projectional bone mineral density (BMD) and bone mineral content (BMC), we obtained posteroanterior (PA) dual X-ray absorptiometry (DXA), lateral supine DXA (Hologic QDR 2000), and quantitative computed tomography (QCT, GE 9800 scanner) in 260 postmenopausal women enrolled in two trials of treatment for osteoporosis. In 223 women, all vertebral levels, i.e., L2-L4 in the DXA scan and L1-L3 in the QCT scan, could be evaluated. Fifty-five women were diagnosed as having at least one mild fracture (age 67.9 +/- 6.5 years) and 168 women did not have any fractures (age 62.3 +/- 6.9 years). We derived three estimates of "volumetric bone density" from PA DXA (BMAD, BMAD*, and BMD*) and three from paired PA and lateral DXA (WA BMD, WA BMDHol, and eVBMD). While PA BMC and PA BMD were significantly correlated with height (r = 0.49 and r = 0.28) or weight (r = 0.38 and r = 0.37), QCT and the volumetric bone density estimates from paired PA and lateral scans were not (r = -0.083 to r = 0.050). BMAD, BMAD*, and BMD* correlated with weight but not height. The associations with vertebral fracture were stronger for QCT (odds ratio [QR] = 3.17; 95% confidence interval [CI] = 1.90-5.27), eVBMD (OR = 2.87; CI 1.80-4.57), WA BMDHol (OR = 2.86; CI 1.80-4.55) and WA-BMD (OR = 2.77; CI 1.75-4.39) than for BMAD*/BMD* (OR = 2.03; CI 1.32-3.12), BMAD (OR = 1.68; CI 1.14-2.48), lateral BMD (OR = 1.88; CI 1.28-2.77), standard PA BMD (OR = 1.47; CI 1.02-2.13) or PA BMC (OR = 1.22; CI 0.86-1.74). The areas under the receiver operating characteristic (ROC) curves for QCT and all estimates of volumetric BMD were significantly higher compared with standard PA BMD and PA BMC. We conclude that, like QCT, estimates of volumetric bone density from paired PA and lateral scans are unaffected by height and weight and are more strongly associated with vertebral fracture than standard PA BMD or BMC, or estimates of volumetric density that are solely based on PA DXA scans.

Absorptiometry, Photon↗
PubMed1995

Analysis of copper-induced metallothionein expression using autonomously replicating plasmids in Candida glabrata.

Candida glabrata strains and a stable plasmid were developed that were suitable for analysis of copper-inducible expression from promoters of the three metallothionein (MT) genes. The two homologous MTII genes, MTIIa and MTIIb, encode the same polypeptide but are differentially induced by copper salts. MTIIb is more highly inducible than MTIIa and cells harboring a single MTIIb exhibit a greater resistance to copper salts compared to cells harboring a single MTIIa. The differential copper inducibility was mapped to sequences between -03 and -292 upstream of the MT coding sequences. Expression of MTI is highly Cu-regulated, but this MT gene confers much less resistance than MTII genes.

Base Sequence↗
PubMed1995

Novel insertion sequence-like element IS982 in lactococci.

A novel insertion sequence-like (IS) element, designated IS982, was found on the lactose plasmid, pSK11L, from Lactococcus lactis subsp. cremoris SK11 and was located between the origin of replication and the oligopeptide transport gene cluster. The 1003-base pair (bp) IS982 was flanked by 18-bp perfect inverted repeats. IS982 contained an open reading frame encoding a putative transposase of 296 amino acids. An almost identical IS-like element (99% DNA sequence identity) was cloned and partially sequenced from the chromosome of Lactococcus lactis subsp. cremoris Wg2 with 17-bp perfect inverted repeats. Southern analysis indicated that in 12 lactococcal strains examined, IS982 was present with copy numbers ranging from 1 to at least 20. IS982 displayed sequence homology to the putative IS element RSBst-alpha from Bacillus stearothermophilus CU21, IS982 and RSBst-alpha were not related to other known insertion sequences and may represent a new family of IS elements.

Amino Acid Sequence↗
PubMed1995

Lactococcin A overexpression in a Lactococcus lactis subsp. lactis transformant containing a Tn5 insertion in the lcnD gene.

Lactococcin A production in lactococci has recently been linked to a signal-sequence-independent secretory system consisting of a four-gene cluster. Lactococcus lactis subsp. lactis LLM23L-A1 has been obtained after Tn5 mutagenesis of pLLM23, a plasmid containing the gene cluster responsible for lactococcin A production. In contrast to other Tn5-generated mutants, strain LLM23L-Al exhibited a 12-fold increase in lactococcin A production. Overproduction of lactococcin A was not linked to an increased pLLM23 copy number. Restriction-enzyme analysis indicated the site of Tn5 insertion to be at the 3' end of lcnD, and upstream of the lcnA structural gene. From DNA sequencing, the Tn5 insertion was located -79 bp upstream of the transcription start site of the lcnA and lciA genes, eliminating eight amino acids from the C-terminal end of lactococcin D. Northern blots revealed overproduction of a 500-base transcript in strain LLM23L-A1, which corresponded to that predicted from the positions of the lactococcin A operon transcriptional start site and the termination structures. This result suggests that the overproduction of lactococcin A in strain LLM23L-A1 is at the transcriptional level and provides further impetus for elucidating the complete regulatory mechanism for lactococcin A expression.

Bacterial Proteins↗
PubMed1995

Influence of degenerative joint disease on spinal bone mineral measurements in postmenopausal women.

We assessed the impact of various forms of spinal degenerative joint disease (DJD) on bone mineral density (BMD) measured by quantitative computed tomography (QCT) and dual X-ray absorptiometry (DXA) in a group of postmenopausal women. Lateral (T4-L4) and AP (L1-L4) spinal radiographs were reviewed for fracture and DJD in 209 women (mean age 62.6 +/- 6.7). The severity of DJD findings was graded as 0, 1, or 2 on the lumbar films, except for vertebral osteophytes which were graded from 0 to 3. Vertebral fractures were defined semiquantitatively as approximately 20% reduction in anterior, middle, or posterior vertebral height. BMD was measured in all subjects by QCT and DXA, including posteroanterior DXA (PA-DXA), lateral DXA (L-DXA) and midlateral DXA (mL-DXA). When BMD was measured by QCT and mL-DXA in the 168 women without fractures, no significant differences were found between women with and those without DJD. However, BMD by PA-DXA was significantly higher in women with DJD changes, particularly when osteophytes were present at the vertebral bodies or facet joints. BMD by L-DXA was less affected by DJD. For this measurement a significant increase in BMD was only noted in subjects with vertebral osteophytes. Multivariate analysis of variance (MANOVA) showed that BMD by QCT and mL-DXA was not affected by DJD. In contrast, for all women, BMD by PA- and L-DXA was affected more by DJD than by fracture status. Chi-square testing demonstrated no significant relationships between vertebral fractures and any of the DJD changes. We conclude that QCT and mL-DXA are superior to PA-DXA and L-DXA in detecting bone loss in patients with DJD. Thus, for these patients, BMD assessment by QCT or mL-DXA may be advisable.

Absorptiometry, Photon↗
PubMed1995

Spinal bone mineral assessment in postmenopausal women: a comparison between dual X-ray absorptiometry and quantitative computed tomography.

We compared quantitative computed tomography (QCT) and dual X-ray absorptiometry (DXA) with respect to their ability to discriminate subjects with and without prevalent vertebral fractures. In 240 post-menopausal women (mean age 63.7 +/- 6.9 years) lateral spine radiographs (T4-L4) were reviewed for the presence of vertebral fracture. Using a semiquantitative technique to grade the severity of vertebral deformities, we classified fractures as mild, moderate or severe (grade 1 to 3, respectively). Postero-anterior DXA (PA-DXA) and lateral DXA (L-DXA) measurements (L2-4) as well as QCT measurements of the lumbar spine (T12-L3 or L1-14) were obtained in all women. Seventy-two women were diagnosed with at least one fracture, and of these 40 were graded as mild. Comparing normal women with fractured women, we found the area under the receiver operating characteristics (ROC) curves to be greatest for QCT (0.81), followed by L-DXA (0.72) and PA-DXA (0.65). The differences among all three techniques were significant. Comparing the normal women with women having only mild fractures, the areas under the ROC curves were 0.79, 0.73 and 0.63 for QCT, L-DXA and PA-DXA, respectively. Significant differences existed between QCT and PA-DXA as well as between L-DXA and PA-DXA. Logistic regression analysis also revealed the highest age-adjusted odds ratios for QCT (3.67; 2.25-5.97) while L-DXA and PA-DXA showed substantially lower odds ratios (2.00; 1.39-2.87, and 1.54; 1.11-2.15, respectively). We conclude that low bone density as measured by QCT, PA-DXA or L-DXA is significantly associated with the prevalence of vertebral fractures. Of the methods studied, QCT of trabecular bone offered the best discriminatory capability. L-DXA proved to be superior to PA-DXA in its diagnostic sensitivity, particularly in women with mild fracture. Mild vertebral fractures are associated with decreased spinal bone density and may be regarded as osteoporotic deformities.

Absorptiometry, Photon↗
PubMed1995

Hyperosmotic activation of transmitter release from presynaptic terminals onto retinal ganglion cells.

A method for evoking neurotransmitter release without light stimulation has been developed and applied to a retinal slice preparation of the tiger salamander (Ambystoma Tigrum). This method utilizes a micropipette containing hyperosmotic levels of sucrose in Ringer, positioned within the inner plexiform layer (IPL) under visual control. Intermittent pressure (between 0.1 and 2 bars) applied to the pipette evoked release of neurotransmitters which were evaluated with whole-cell recording (WCR) technique applied to cells in the ganglion cell layer. Pharmacological studies were used to characterize the properties of the hyperosmotic sucrose-evoked response (HSER) and in some cases, we compared the HSER with synaptic currents evoked by light stimulation. The HSER typically consisted of both inhibitory and excitatory components with a reversal potential in between that for chloride (approximately -60 mV) and non-specific cation channels (approximately 0 mV). Relatively pure inhibition or excitation could be revealed through pharmacological techniques by blocking the inhibition with picrotoxin/strychnine or by blocking the glutamatergic neurotransmission with D-AP7 (D-2-amino-7-phosphonoheptanoate) and NBQX (2,3-dihydroxy-6-nitro-sulfamoyl -benzo (F) quinoxaline). A comparison of light-evoked responses (LER) and the HSER suggested that they activate the same pool of releasable neurotransmitter.

2-Amino-5-phosphonovalerate↗
PubMed1995

RNA editing in higher plant mitochondria: analysis of biochemistry and specificity.

RNA editing alters genomically encoded cytidines to uridines posttranscriptionally in higher plant mitochondria. Most of these editing events occur in translated regions and consequently alter the amino acid sequence. In Oenothera berteriana more than 500 editing sites have been detected and the total number of editing sites exceeds 1000 sites in this mitochondrial genome. To identify the components involved in this process we investigated the factors determining the specificity of RNA editing and the apparent conversion of cytidine to uridine residues. The possible biochemical reactions responsible for RNA editing in plant mitochondria are de- or transamination, base substitution and nucleotide replacement. In order to discriminate between these different biochemical mechanisms we followed the fate of the sugar-phosphate backbone by analysing radiolabeled nucleotides after incorporation into high molecular mass RNA. Plant mitochondria were supplied with [alpha-32P]CTP to radiolabel CMP residues in newly synthesized transcripts. Radiolabeled mtRNA was extracted and digested with nuclease P1 to hydrolyse the RNA to monophosphates. The resulting monophosphates were analysed on one- and two-dimensional TLC systems to separate pC from pU. Radiolabeled pU was detected in increasing quantities during the course of incubation. These results suggest that RNA editing in plant mitochondria involves either a deamination or a transglycosylation reaction. The editing product was identified as uridine and not as a hypermodified nucleotide which is recognized as uridine. Similar results have been obtained by incubating in vitro transcribed mRNAs with mitochondrial lysates indicating that RNA editing and transcription is not directly linked in plant mitochondria.(ABSTRACT TRUNCATED AT 250 WORDS)

Biological Evolution↗
PubMed1995

Endoluminal repair of atypical dissecting aneurysm of descending thoracic aorta and fusiform aneurysm of the abdominal aorta.

A 62-year-old male patient was admitted with acute dissociation of the descending thoracic aorta and an infrarenal abdominal aortic aneurysm (AAA). Investigation revealed that the thoracic dissection probably had arisen retrogradely in the posterior wall of the AAA and extended superiorly to the left subclavian artery as a blind sac. Implantation of an endoluminal graft device below the renal arteries enabled simultaneous treatment of the AAA and the thoracic aortic dissection. The patient had an uncomplicated recovery. Postoperative aortography and computed tomography demonstrated normal flow through the aorta and endograft without leak of contrast into the AAA sac or the false lumen of the dissection. Contrast computed tomography 6 months after operation demonstrated that the false lumen was no longer evident.

Aortic Dissection↗
PubMed1995

Early outcome and intermediate follow-up of vascular stents in the femoral and popliteal arteries without long-term anticoagulation.

PURPOSE: The role of arterial stenting in the treatment of femoral and popliteal arterial disease is controversial and has been hampered by recommendations for patients to be given anticoagulants (oral warfarin) for several months or more. This study was undertaken to evaluate the immediate and midterm outcomes of vascular stents implanted percutaneously in the femoral and popliteal arteries, without long-term anticoagulation. METHODS: Over a 3-year period, 32 patients admitted to a vascular surgery service had arterial stents implanted in the femoral (n = 22) or popliteal (n = 10) artery for the following indications: recurrent stenosis after angioplasty (n = 13), suboptimal result after angioplasty of occluded (n = 12) or calcified stenotic arteries (n = 2), percutaneous transluminal angioplasty-induced thrombosis or dissection (n = 5). Access to the artery was gained by percutaneous insertion of a hemostatic sheath into the ipsilateral common femoral artery. Systemic heparin was given at the time of stent insertion, and patients were prescribed daily low-dose aspirin. RESULTS: Successful stent implantation was achieved in 31 of the 32 patients. Acute thrombosis (< 30 days) occurred in two patients. There was no incidence of false aneurysm formation, acute leg ischemia, or vessel perforation. All patients were monitored by Doppler scanning index and duplex scanning within 24 hours, and thereafter at 3- to 6-month intervals. The mean ankle-brachial systolic pressure index improved from 0.60 (before treatment) to 0.88 (3 to 6 months after stenting). Stent occlusion has occurred in six patients; two stents were successfully salvaged with urokinase infusion. In follow-up to date (range 3 to 33 months) the primary patency rate by life-table analysis was 75% at 18 months, whereas the secondary patency rate was 89% at the same interval. Restenosis (> 50% lumen diameter) was detected by duplex ultrasonography in seven of 25 patent stents (28%) at a mean interval of 9.5 months (range 4 to 15 months); of these, four patients remained clinically symptom-free despite the ultrasound findings. CONCLUSIONS: We conclude that vascular stents can be implanted into the femoropopliteal arteries with few complications and with acceptable early and intermediate patency rates, without the need for long-term anticoagulation. Restenosis is not prevented by stents, and the main value of stenting at this site appears to be in salvaging acute complications of percutaneous transluminal angioplasty, or to correct suboptimal results after recanalization of occluded arteries.

Aged↗
PubMed1995

Surgical management of complications following endoluminal grafting of abdominal aortic aneurysms.

OBJECTIVE: The aim of this study was to report the outcome of endoluminal grafting of abdominal aortic aneurysms (AAA) with special reference to complications. METHODS: Between May 1992 and August 1994 endoluminal repair of aneurysms was undertaken in 61 patients. In 53 the aneurysm was aortic and these are the basis of this report. In patients with AAA all procedures were elective and were performed in the operating room with the patient draped for an open repair in the event of failed endoluminal repair. The configuration of the endografts was tubular 36, tapered aortoiliac/aortofemoral 12 and bifurcated 5. Radiographic guidance was used to pass the endografts into the aorta via a delivery sheath introduced through the femoral or iliac arteries. RESULTS: Successful endoluminal repair of AAA was achieved in 43 of 53(81%) patients. In the remaining 10 patients, endoluminal repair was abandoned in favour of an open repair. There were 17(32%) local/vascular and 13(25%) systemic/remote remote complications. The sum of these complications occurring in successful endoluminal repairs and those complications leading to failure of endoluminal repair was 40(75%). There were two cardiac deaths within 30 days in patients undergoing endoluminal repair (both procedure related) and four late deaths (unrelated to aneurysm repair). Three of the late deaths were in patients undergoing endoluminal repair and one endoluminal converted to open repair. CONCLUSION: Endoluminal repair of AAA in our experience has a low perioperative (< 30 days) mortality rate (3.7%) but a high morbidity rate (75%). It is recommended that complications be classified into three groups: systemic/remote and local/vascular (following successful endoluminal repair) plus those complications leading to failure of endoluminal repair. The first group is composed of medical complications while the latter two groups comprise those surgical complications directly related to the endoluminal technique.

Aged↗
PubMed1995

RRR-alpha-tocopheryl succinate inhibits DNA synthesis and enhances the production and secretion of biologically active transforming growth factor-beta by avian retrovirus-transformed lymphoid cells.

The RRR-alpha-tocopheryl succinate form of vitamin E, referred to as vitamin E succinate (VES), inhibits the proliferation of avian reticuloendotheliosis virus-transformed RECC-UTC4-1 (C4-1) lymphoblastoid cells in a dose-dependent manner in vitro. Analyses of conditioned medium (CM) from VES growth-inhibited cells revealed a potent antiproliferative activity. Characterization of the antiproliferative activity as transforming growth factor-beta (TGF-beta) was established by 1) growth inhibition of TGF-beta-responsive Mv1Lu mink lung and murine CTLL-2 cell lines, 2) a combination of physical characteristics including heat stability, acid stability, and Bio-Gel P-60 column chromatography elution profile, 3) neutralization of the antiproliferative activity by antibodies specific for TGF-beta, and 4) immunoprecipitation of metabolically labeled TGF-beta in CM from VES-treated C4-1 cells by use of TGF-beta-specific antibodies. Northern blot analyses of total cellular RNA revealed that VES does not alter the levels of constitutively expressed TGF-beta isoform-specific mRNAs; namely, VES does not alter the levels of the 3.9- and 4.1-kb TGF-beta 2 mRNAs, the 3.0-kb TGF-beta 3 mRNA, or the 2.5-, 2.7-, and 1.7-kb TGF-beta 4 mRNAs. The data show that VES inhibits C4-1 cell proliferation and induces the cells to produce and secrete active forms of TGF-beta, suggesting that one mechanism whereby VES inhibits C4-1 cell proliferation may be via the TGF-beta pathway for cellular growth control.

Animals↗
PubMed1995

Transport of dendritic microtubules establishes their nonuniform polarity orientation.

The immature processes that give rise to both axons and dendrites contain microtubules (MTs) that are uniformly oriented with their plus-ends distal to the cell body, and this pattern is preserved in the developing axon. In contrast, developing dendrites gradually acquire nonuniform MT polarity orientation due to the addition of a subpopulation of oppositely oriented MTs (Baas, P. W., M. M. Black, and G. A. Banker. 1989. J. Cell Biol. 109:3085-3094). In theory, these minus-end-distal MTs could be locally nucleated and assembled within the dendrite itself, or could be transported into the dendrite after their nucleation within the cell body. To distinguish between these possibilities, we exposed cultured hippocampal neurons to nanomolar levels of vinblastine after one of the immature processes had developed into the axon but before the others had become dendrites. At these levels, vinblastine acts as a kinetic stabilizer of MTs, inhibiting further assembly while not substantially depolymerizing existing MTs. This treatment did not abolish dendritic differentiation, which occurred in timely fashion over the next two to three days. The resulting dendrites were flatter and shorter than controls, but were identifiable by their ultrastructure, chemical composition, and thickened tapering morphology. The growth of these dendrites was accompanied by a diminution of MTs from the cell body, indicating a net transfer of MTs from one compartment into the other. During this time, minus-end-distal microtubules arose in the experimental dendrites, indicating that new MT assembly is not required for the acquisition of nonuniform MT polarity orientation in the dendrite. Minus-end-distal microtubules predominated in the more proximal region of experimental dendrites, indicating that most of the MTs at this stage of development are transported into the dendrite with their minus-ends leading. These observations indicate that transport of MTs from the cell body is an essential feature of dendritic development, and that this transport establishes the nonuniform polarity orientation of MTs in the dendrite.

Animals↗
PubMed1995

Optimal staining and sample storage time for direct microscopic enumeration of total and active bacteria in soil with two fluorescent dyes.

Direct counting techniques, first developed for aquatic samples, can be used to enumerate bacteria in soil and groundwater sediments. Two fluorescent dyes, 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) for actively respiring bacteria and 4(prm1),6-diamidino-2-phenylindole (DAPI) for total bacteria, were tested for their usefulness in epifluorescent direct bacterial enumeration in soil. Both dyes can be used for the same soil sample without affecting enumeration results. Staining for 8 h with CTC and for 40 min with DAPI resulted in maximum numbers of stained cells. The optimal DAPI staining concentration is 10 mg liter(sup-1). After preparation, slides should be stored at 4(deg)C and counted within 2 days for CTC and within 24 h for DAPI. Sodium PP(infi) or sodium chloride solutions were used to desorb bacteria from soil prior to counting. Counts were significantly higher when sodium chloride was used.

Journal Article↗
PubMed1995

Specific binding of host cellular proteins to multiple sites within the 3' end of mouse hepatitis virus genomic RNA.

The initial step in mouse hepatitis virus (MHV) RNA replication is the synthesis of negative-strand RNA from a positive-strand genomic RNA template. Our approach to begin studying MHV RNA replication is to identify the cis-acting signals for RNA synthesis and the proteins which recognize these signals at the 3' end of genomic RNA of MHV. To determine whether host cellular and/or viral proteins interact with the 3' end of the coronavirus genome, an RNase T1 protection/gel mobility shift electrophoresis assay was used to examine cytoplasmic extracts from mock- and MHV-JHM-infected 17Cl-1 murine cells for the ability to form complexes with defined regions of the genomic RNA. We demonstrated the specific binding of host cell proteins to multiple sites within the 3' end of MHV-JHM genomic RNA. By using a set of RNA probes with deletions at either the 5' or 3' end or both ends, two distinct binding sites were located. The first protein-binding element was mapped in the 3'-most 42 nucleotides of the genomic RNA [3' (+42) RNA], and the second element was mapped within an 86-nucleotide sequence encompassing nucleotides 171 to 85 from the 3' end of the genome (171-85 RNA). A single potential stem-loop structure is predicted for the 3' (+)42 RNA, and two stem-loop structures are predicted for the 171-85 RNA. Proteins interacting with these two elements were identified by UV-induced covalent cross-linking to labeled RNAs followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The RNA-protein complex formed with the 3'-most 42 nucleotides contains approximately five host polypeptides, a highly labeled protein of 120 kDa and four minor species with sizes of 103, 81, 70, and 55 kDa. The second protein-binding element, contained within a probe representing nucleotides 487 to 85 from the 3' end of the genome, also appears to bind five host polypeptides, 142, 120, 100, 55, and 33 kDa in size, with the 120-kDa protein being the most abundant. The RNA-protein complexes observed with MHV-infected cells in both RNase protection/gel mobility shift and UV cross-linking assays were identical to those observed with uninfected cells. The possible involvement of the interaction of host proteins with the viral genome during MHV replication is discussed.

Animals↗