Coamplification of HIV-1 proviral DNA and viral RNA in assays used for quantification of HIV-1 RNA.

Elevated HIV-1 viral load (VL) observed in specimens frozen in situ in plasma preparation tubes (PPTs) compared to EDTA plasma specimens may affect therapeutic monitoring of HIV-infected patients. The increase in viral load is cell associated and minimized when plasma from the PPT is aspirated or recentrifuged prior to freezing. This study investigates the contribution of integrated HIV-1 proviral DNA to elevated VL in the quantification of HIV-1 RNA in plasma. Fifty paired specimens collected in EDTA tubes and PPTs frozen in situ were used for analysis. HIV-1 VL was measured using the COBAS Amplicor Monitor ultrasensitive test version 1.5. Contaminating proviral DNA was detected using a nested PCR targeting the Alu repeat in human genomic DNA and HIV pol gene simultaneously. Treatment of the specimen with DNase resulted in significantly lower quantifiable HIV-1 RNA in specimens from PPTs compared to the corresponding EDTA tubes (P = 0.004). After the RNA was destroyed by heat treatment, the mean HIV-1 RNA VL decreased by 79% in the EDTA tube compared to 65% in the PPT. The nested PCR amplified integrated proviral DNA in nucleic acid extracted from plasma in PPT and EDTA specimens with high viral load values. Likewise, a semiquantitative densitometric analysis revealed that the total amount of genomic DNA in the PPT was higher than that in the EDTA tube. Our investigation clearly shows that both proviral DNA and intracellular RNA are amplified simultaneously in the COBAS Amplicor HIV-1 Monitor assay and that proviral DNA contributes to the elevated VL in plasma frozen in PPTs.

Circulating nucleic acids of Chlamydia pneumoniae and cytomegalovirus in patients undergoing coronary angiography.

Peripheral blood mononuclear cells from 208 consecutive patients undergoing elective coronary angiography or angioplasty were collected before, immediately after, and 4 h after the procedure. Nucleic acids of Chlamydia pneumoniae and of cytomegalovirus (CMV) were detected by PCR and confirmed by hybridization. Circulating C. pneumoniae DNA was identified in 24 patients (11.5%) and was associated with current smoking (odds ratio [OR] = 4.5, 95% confidence interval [CI] = 1.6 to 12.2, P = 0.004) but not with arterial narrowing on coronary angiogram or with serological results positive for C. pneumoniae. Circulating CMV DNA was identified in 36 patients (17.3%) and was associated with anti-CMV immunoglobulin G (OR = 2.7, 95% CI = 1.2 to 6.3, P = 0.02) but not with angiographic arterial narrowing or with the need for revascularization. Neither C. pneumoniae nor CMV DNA detection increased after angioplasty, a procedure in which endothelium is disrupted. Larger prospective studies are needed to determine the prognostic significance of DNA detection.

Stability of plasma human immunodeficiency virus load in VACUTAINER PPT plasma preparation tubes during overnight shipment.

VACUTAINER PPT plasma preparation tubes were evaluated to determine the effects of various handling and shipping conditions on plasma human immunodeficiency virus (HIV) load determinations. Plasmas obtained from PPT tubes stored and shipped under nine different conditions were compared to conventional EDTA tube plasmas stored at -70 degrees C within 2 h after phlebotomy. Compared to viral loads in frozen EDTA plasma, those in PPT tube plasma that was frozen immediately and either separated or shipped in situ were not significantly different. Viral loads in PPT tube plasma after storage for 6 h at either room temperature or 4 degrees C, followed by shipment at ambient temperature or on wet or dry ice, were not significantly different from baseline viral loads in EDTA or PPT plasma. The results of this study indicate that the HIV load in PPT tube plasma is equivalent to that in standard EDTA plasma. Plasma viral load is not affected by storage or shipment temperature when plasma is collected in PPT tubes. Furthermore, plasmas can be shipped in spun PPT tubes, and the tubes provide a safer and more convenient method for sample collection and transport than regular EDTA tubes.

Antibodies distinguishing between intact and alkali-hydrolyzed 7-methylguanosine.

Antibodies specific for intact 7-methylguanosine (m7G) were induced in rabbits and mice by immunization with nucleoside-BSA or nucleoside-hemocyanin conjugates. Since m7G undergoes alkali-catalyzed hydrolytic fission of the purine ring, modifications were made in the procedure for conjugation of m7G to proteins. After periodate oxidation, m7G was incubated with protein at pH 9.1 at 4 degrees C for one hour during which the nucleoside was found to be stable. Reduction of the Schiff base was done with t-butylamine borane for 30 minutes, and the conjugated protein was isolated quickly by gel filtration at pH 7.2. Both rabbits and mice produced antibodies that readily distinguished between the intact and hydrolyzed m7G. Antibody specificity depended largely on the presence of an intact 7-substituted imidazole ring and some cross-reaction occurred with 7-methylinosine. A weaker reaction occurred with ribothymidine and thymidine. Mouse antibodies induced by m7G-hemocyanin showed the highest specificity. They also recognized m7G in the isolated mRNA cap structure m7G(5')ppp(5')A.

Antisera to poly(A)-poly(U)-poly(I) contain antibody subpopulations specific for different aspects of the triple helix.

Rabbit antibodies to the triple-helical polynucleotide poly(A)-poly(U)-poly(I) were fractionated into three major antibody populations, each recognizing a different conformational feature of the triple-helical immunogen. Two distinct populations were purified from precipitates made with poly(A)-poly(U)-poly(U) and poly(A)-poly(I)-poly(I). The former reacted with double-stranded poly(A)-poly(U) or poly(I)-poly(C), and similar populations could be purified with either double-stranded form. The second population recognized the poly(A)-poly(I) region of the triple helix, and the third required all three strands for reactivity. These immunochemical studies suggest that the poly(A) and poly(U) have the same orientation in the triple-helicical poly(A)-poly(U)-poly(I) as in the double-helical poly(A)-poly(U), in which they have Watson-Crick base pairing.

Interaction of Rhodium(II) carboxylates with molecules of biologic importance.

Rhodium(II) acetate, propionate, and butyrate showed a considerable variation in their antitumor activity against Ehrlich ascites tumor cells in mice, with the butyrate complex being the most active. The three complexes markedly inhibited DNA synthesis of Ehrlich ascites tumor cells in vivo. Rhodium (II) butyrate was the most potent inhibitor followed by the propionate complex. One hour after administration, rhodium(II) propionate and butyrate induce more uridine-5-3H incorporation into RNA than is seen in the controls. Equilibrium dialysis studied showed that rhodium(II) acetate-1-14C binds to single stranded DNA, poly-A, ribonuclease A, and bovine serum albumin but not to highly polymerized native calf thymus DNA, poly-G, or poly-C. In these cases binding occurred at the two axial positions of rhodium(II) acetate to a nitrogen donor in the ligands. The formation constants of the rhodium(II) acetate and propionate complexes with 5'-adenosine monophosphate were determined. The rhodium(II) propionate complex was more stable. Sedimentation and viscosity measurements of poly-A and poly-A/rhodium(II) acetate complexes indicate a high degree of intramolecular crosslinking in the rhodium(II) acetate/poly-A complex. The rhodium(II) carboxylate complexes were also found to be potent inhibitors of purified DNA polymerase I and RNA polymerase from Escherichia coli.