Replication of heterologous combinations of helper and defective RNA of citrus tristeza virus

Citrus tristeza virus (CTV) populations are among the more complex of plant RNA viruses with unusual mixtures of strains and defective RNAs (dRNAs). Citrus plants infected with different CTV isolates contain multiple dRNA molecules that differ in size and relative abundance within and between isolates. Additionally, we found mixtures of heterologous dRNAs in populations. To examine the replication of CTV dRNAs, the protoplast system had to be extended to support helper-assisted amplification of input dRNAs. The use of freshly extracted sap of CTV-infected tissue as inoculum increased the infection of Nicotiana benthamiana protoplasts sufficiently to result in accumulation of high levels of CTV RNAs as well as dRNAs within 2 or 3 days postinoculation. A series of dRNA-like molecules, each with a single large internal deletion, were created from an infectious cDNA clone of the CTV T36 isolate and examined for amplification in N. benthamiana protoplasts using a CTV deletion mutant as the helper virus. Of 12 synthetic dRNAs, only three with sizes of 3650, 3819, and 4460 nucleotides were efficiently replicated. CTV dRNA replication did not appreciably affect levels of accumulation of the genomic or the subgenomic RNAs of the helper virus. To investigate the maintenance of dRNAs in CTV populations, we examined heterologous interactions between dRNAs and helper viruses. Wild-type populations of heterologous strains T68 and T3, as well as the homologous T36, supported replication of synthetic T36 dRNAs. Replacement in the T36 dRNA of the 5' region, which is most variable among CTV strains, with the corresponding sequences from VT, T68, T3, or T30 resulted in chimeric dRNAs that failed to be replicated by the T36 helpers but were replicated to detectable levels by the T68 helper. The differential specificities of different CTV replicase complexes with dRNA replication signals is one possible factor that affects the maintenance of dRNA population structures.     

Preclosure Pressure Gradients Predict Patent Ductus Arteriosus Patients at Risk for Later Left Pulmonary Artery Stenosis

The objective of this study was to evaluate the incidence of pre-existing catheterization left pulmonary artery (LPA) gradients and correlation of these gradients with later LPA stenosis after successful patent ductus arteriosus (PDA) occlusion. We performed a single-center review of 130 patients with PDA closure from October 1993 to February 2005. We analyzed the pre-PDA closure LPA pressure gradients at catheterization to determine if these were predictive of late LPA stenosis. On follow-up, a V _max >2 m/s by echocardiogram (transthoracic echocardiography; TTE) was considered indicative of possible LPA stenosis. Left lung perfusion of <35% was considered diagnostic of significant LPA stenosis. Post PDA closure, possible LPA stenosis by TTE was seen in 8 of 128 patients (6.25%). Seven of these eight had precatheter LPA gradients >7 mm Hg. Five of these had perfusion scans, three of the five had significant LPA stenosis, and two underwent LPA angioplasty. Patients with LPA catheter gradients >7 mm Hg were more likely to have possible LPA stenosis by TTE, significant LPA stenosis by lung scan, and intervention with LPA angioplasty. In conclusion, a preclosure main pulmonary artery-to-LPA pressure gradient >7 mm Hg was found in all patients who developed significant LPA stenosis on follow-up after transcatheter PDA closure. It appears likely that these patients have LPA abnormality rather than stenosis caused by the PDA occlusion device. Patients with preclosure LPA gradients >7 mm Hg should undergo follow-up evaluations for detection of significant stenosis and may require treatment if an important flow abnormality is documented.     

Can optical coherence tomography predict the outcome of laser photocoagulation for diabetic macular edema?

BACKGROUND AND OBJECTIVE: To assess the outcome of laser photocoagulation in patients with diabetic macular edema. PATIENTS AND METHODS: Forty-seven patients (51 eyes) with clinically significant macular edema (CSME) undergoing grid laser photocoagulation were included. Clinical examination and optical coherence tomography (OCT) were performed at baseline and 3 to 4 months after treatment. The central foveal thickness, mean inner macular thickness (average retinal thickness in fovea and inner macular circle), and mean macular thickness were calculated. Based on the greatest OCT thickness at baseline, patients were grouped according to mild (< 300 microm; Group 1), moderate (300 to 399 microm; Group 2), and severe (> or = 400 microm; Group 3) macular edema. RESULTS: Group 2 showed significant reductions in central foveal thickness (23 microm, P = .02), mean inner macular thickness (18 microm, P = .02), and mean macular thickness (9 microm, P = .04) with slight improvement in visual acuity. Groups 1 and 3 did not show any significant change in macular thickness values and there was a statistically insignificant worsening of visual acuity in these groups. CONCLUSIONS: Patients with moderate macular thickening of 300 to 400 microm benefit most from laser treatment. OCT may help in choosing the appropriate treatment for CSME based on the degree of macular thickening. Long-term studies are warranted to confirm these findings.     

Origin of left main and right coronary arteries from right aortic sinus of Valsalva

Coronary anomalies may be a part of complex congenital malformations of the heart or be an isolated defect. Anomalous coronary arteries are associated with a higher incidence of congenital heart diseases, but do not appear to be associated with an increased risk for development of coronary atherosclerosis. Coronary anomalies are recognized readily on angiography. Unexpected findings during invasive procedures would suggest a possibly existing coronary anomaly, especially when main branches cannot be opacified by selective contrast medium injection. This case report illustrates the clinical and angiographic findings of a patient undergoing coronary angiography for evaluation of ischemic heart disease with an unexpected presence of anomalous origin of the left coronary artery from the right aortic sinus.     

Anticoagulant effect of Naja naja venom 5'nucleotidase: demonstration through the use of novel specific inhibitor, vanillic acid.

The snake venom proteins affect hemostasis by either advancing/delaying blood coagulation. Apart from proteases and phospholipase A(2)s (PLA(2)s), 5'nucleotidase is known to affect hemostasis by inhibiting platelet aggregation. In this study, the possible involvement of Naja naja venom 5'nucleotidase in mediating anticoagulant affect is evaluated. Vanillic acid selectively and specifically inhibited 5'nucleotidase activity among other enzymes present in N. naja venom. It is a competitive inhibitor as evident of inhibition relieving upon increased substrate concentration. Vanillic acid dose dependently inhibited the anticoagulant effect of N. naja venom up to 40%. This partial involvement of 5'nucleotidase in mediating anticoagulant effect is substantiated by concanavalin-A (Con-A) inhibition studies. Con-A, competitively inhibited in vitro protease and 5'nucleotidase activity up to 100%. However, it did not exhibit inhibitory activity on PLA(2). The complete inhibition of anticoagulant effect by Con-A upon recalcification time suggests the participation of both 5'nucleotidase and protease in mediating anticoagulant effect of N. naja venom. Vanillic acid and Con-A inhibition studies together suggest that probably 5'nucleotidase interacts with one or more factors of intrinsic pathway of blood coagulation to bring about anticoagulant effect. Thus, this study for the first time demonstrates the involvement of 5'nucleotidase in mediating N. naja venom anticoagulant effect.     

The p20 gene product of Citrus tristeza virus accumulates in the amorphous inclusion bodies

Citrus tristeza virus (CTV) has 10 3' open reading frames (ORFs) of unknown function except for the two coat proteins. The highest produced subgenomic RNAs are those of the major coat protein gene (p25) and the 3' most genes, p20 and p23. The proteins from three ORFs, p25, p27, and p20, were examined in the yeast two-hybrid assay for the interactions between themselves and to one another. The p20 protein exhibited a high affinity for itself, suggesting that it might aggregate in infected cells. The cytopathology of CTV infections includes characteristic paracrystalline and amorphous inclusions in the phloem elements of infected citrus. Polyclonal antiserum raised against the bacterial expressed p20 gene product detected a protein of approximately 22-23 kDa, which accumulated to relatively high levels in CTV-infected citrus, but not in healthy citrus. Immunogold localization using antibodies to p20 protein showed strong and specific labeling of the amorphous inclusion bodies present in CTV-infected cells. Mesophyll protoplasts of Nicotiana benthamiana transfected with a CTV mutant containing the green fluorescent protein (GFP) ORF fused in-frame to the 3' end of p20 protein ORF expressed high levels of GFP. The fusion protein was concentrated in one specific area in the cytoplasm and lacked an organized shape. Accumulation of high levels of p20 protein in infected tissue, specific localization of the p20-GFP fusion protein, immunolocalization of p20 protein into amorphous inclusions, and strong homologous p20 protein-p20 protein interactions in the yeast-two-hybrid assay suggest that the p20 protein of CTV is a major component of the amorphous inclusion bodies present in CTV-infected cells.     

The fitness of citrus tristeza virus defective RNAs is affected by the lengths of their 5'- and 3'-termini and by the coding capacity

Populations of the Closterovirus Citrus tristeza virus (CTV) generally contain defective RNAs (dRNAs) that vary in size, abundance, and sequence. The variation in abundance of the different dRNAs in a population suggests selection for those of higher fitness. To examine factors affecting fitness of dRNAs, we investigated a series of in vitro constructed dRNAs for their ability to be amplified in protoplasts by an efficiently replicated CTV deletion mutant. The minimal sequences required for accumulation of the dRNAs were within the genomic 5' proximal approximately 1 kb and the 3' 270 nucleotides. However, other factors were involved, because a dRNA with only the minimal sequences failed to be replicated. Rescue of a nonviable dRNA by insertion of nonviral sequences between the termini suggested that "spacing" between terminal cis-acting signals influenced fitness. A continuous open reading frame (ORF) through most of the sequences derived from the 5' of the genome was a requirement for dRNA amplification. In general, insertions, deletions, or nucleotide substitutions were tolerated in the dRNAs as long as an ORF was retained, whereas dRNAs with mutations that prematurely terminated the ORF were not viable. To discriminate between a requirement for an essential protein and ribosomal travel, perhaps to present replication signals to the replicase complex, mutations were made to modify the potential protein but still maintain an ORF. Deletions, insertions of nonviral sequences, or switching of reading frames that altered the amino acid sequence of the protein, except the N-terminal 161 amino acids, did not destroy the fitness of the dRNAs. Yet termination of the ORF in the middle of nonviral sequences did destroy the ability of the dRNAs to be amplified. These results suggest that even though a continuous ORF was needed for fitness, its protein product did not affect the amplification of the dRNAs.