Detection of human immunodeficiency virus type 1 DNA by polymerase chain reaction amplification and capture hybridization in microtiter wells.

We developed an improved microtiter-based assay for the detection of polymerase chain reaction (PCR)-amplified DNA sequences. The synthetic DNA sequences used to prime the PCR were labeled with biotin at their 5' ends so that the specific PCR product was labeled with biotin. Following amplification, an aliquot of the PCR product was denatured and hybridized to a capture DNA sequence immobilized in a microtiter well. The capture sequence was complementary to a portion of the sequence between the primers, so that only extended primers were captured. The captured PCR product was detected colorimetrically by using a streptavidin-peroxidase conjugate and tetramethylbenzidine substrate.

Detection of hepatitis B virus DNA in serum by polymerase chain reaction amplification and microtiter sandwich hybridization.

We have developed a microtiter sandwich hybridization assay for the detection of polymerase chain reaction (PCR)-amplified hepatitis B virus (HBV) sequences. This assay utilizes an enzyme-linked immunosorbent assay-like format in which cloned DNA containing a sequence complementary to half of one PCR product strand is immobilized in microtiter wells. A biotin-labeled DNA sequence complementary to the other portion of the same PCR product strand is used as the probe. The DNAs from 69 hepatitis B surface antigen-positive serum samples and 16 antigen-negative control samples were amplified by the PCR procedure, and the product was detected by Southern and sandwich hybridization. Both detection procedures were capable of detecting as few as five copies of HBV DNA. Compared with Southern hybridization, the sandwich hybridization assay exhibited a sensitivity of 100% and a specificity of 95% for the detection of amplified HBV sequences. Unlike Southern hybridization, however, the sandwich hybridization assay employs a nonradioactive probe and allows easy handling of large numbers of samples. DNA was detected in 74% of the antigen-positive samples. All of the antigen-negative samples (healthy blood donors) were negative for HBV DNA by both procedures.

Labeling of DNA probes with a photoactivatable hapten.

A photoactivatable reagent for introducing haptens onto DNA probes has been prepared using a commercially available bifunctional linker arm reagent and amino-derivatized 2,4-dinitrophenyl (DNP). The resulting compound (photo-DNP) couples efficiently to DNA using an ordinary sunlamp. Under optimum conditions, about 7-23 DNP molecules per 1000 bases are incorporated into the DNA. Hybridization experiments demonstrate that as little as 1.5 x 10(5) copies of target DNA can be detected by filter hybridization with a photo-DNP-labeled probe and immunochemical detection.

A sensitive nonisotopic hybridization assay for HIV-1 DNA.

We have developed a microtiter-based sandwich hybridization assay for the detection of low copy number HIV-1 sequences. The assay employs a capture DNA sequence covalently coupled to microtiter wells through linker arms. The detection probe is a biotin-labeled DNA fragment derived from sequences adjacent to the capture sequence. After hybridization in the presence of sample nucleic acid, the detection probe remains bound only if the sample contained complementary sequences spanning the junction between capture and detection probes. The amount of detection probe bound is quantified by incubation with a peroxidase-streptavidin conjugate and a colorimetric peroxidase substrate. This assay has been combined with enzymatic target amplification to achieve sensitive detection of HIV-1 in patient samples. Following amplification of HIV-1 DNA using the polymerase chain reaction technique, a 190-bp product is produced. This product is easily and specifically quantified using the sandwich hybridization assay. The resulting test can detect one HIV-1-infected cell in 10(5) cells or about 30 molecules of HIV-1 DNA.

A chemical method for introducing haptens onto DNA probes.

We have developed a versatile chemical method of attaching hapten moieties onto DNA, for the construction of nonisotopic DNA probes. The DNA is reacted with N-bromosuccinimide at alkaline pH, resulting in bromination of a fraction of the thymine, guanine, and cytosine residues, with adenine modified to a lesser extent. The bromine is subsequently displaced by a primary amino group, attached to a linker arm. The other end of the linker arm has a detectable group preattached to it. We have labeled cloned hepatitis B viral (HBV) DNA with the hapten 2,4-dinitrophenyl (DNP) and used it in combination with a high affinity rabbit anti-DNP antibody, for the detection of hepatitis B DNA by slot blotting. This probe was sensitive enough to specifically detect 1 X 10(-17) mol (1 X 10(6) copies) of HBV DNA in total DNA from human serum.

Induction of collagenase secretion in human fibroblast cultures by growth promoting factors.

In human foreskin fibroblast cultures, two proteins with Mr 60,000 and 55,000 were found to be induced about 3.5-fold by epidermal growth factor (EGF), platelet-derived growth factor, and beta-transforming growth factor. The induced proteins were identified as procollagenases by immunoprecipitation of induced medium with antibodies to purified human fibroblast collagenase. Collagenase enzyme activity in the medium from EGF-treated cultures was also induced at least 3-fold compared to control cultures. Induction of collagenase was dependent upon de novo protein and RNA synthesis and was observed in the medium 10 h after addition of EGF. Although these growth-promoting factors interact with separate membrane receptors, each induced the secretion of a common protein, suggesting that collagenase may be important in some aspect of mitogenesis, cell mobilization, and migration.

5-azacytidine selectively increases gamma-globin synthesis in a patient with beta+ thalassemia.

5-Azacytidine is a cytidine analogue that is capable of activating repressed genes in tissue-culture cells and has been shown to increase hemoglobin-F production in anemic baboons. This drug was administered to a patient with severe beta-thalassemia in an attempt to stimulate hemoglobin-F production. After seven days of 5-azacytidine treatment, gamma-globin synthesis increased approximately sevenfold, temporarily normalizing the patient's unbalanced globin synthesis. Erythropoiesis became more effective, leading to a temporary increase in the absolute reticulocyte count (from 5000 to 22,000 per cubic millimeter) and in hemoglobin concentration (from 8.0 to 10.8 g per deciliter). Hypomethylation of bone-marrow DNA near both the gamma-globin and epsilon-globin genes was directly demonstrated. At the time of peak drug effect, about 7000 gamma-globin messenger RNA molecules were present per erythroid bone-marrow cell, in contrast to 10 to 15 epsilon-globin messenger RNA molecules per cell. 5-Azacytidine selectivity increases gamma-globin synthesis and therefore provides a new approach to the treatment of severe beta-thalassemia. Further studies will be required to evaluate the efficacy, risks, and long-term toxicity of 5-azacytidine (or related compounds) before this approach can be used as a therapy for patients with disorders of hemoglobin synthesis.

Quantitation of phosphatidylcholine secretion in lung slices and primary cultures of rat lung cells.

Rat lung slices and isolated rat lung cells were used to study the secretion of phosphatidylcholine by the lung in vitro. The rate of incorporation of [(3)H]choline by lung slices was 20-fold greater than by liver slices and 4-fold greater in lung cells compared to confluent skin fibroblasts. Labeling lung slices or cells with [(3)H]choline for up to 8 hr failed to reveal a significant amount of labeled phosphatidylcholine in the medium of either system compared to the medium from liver slice or fibroblast controls. Labeling of isolated lung cells for up to 24 hr, with or without 10% fetal calf serum, also showed no significant difference in the amount of labeled phosphatidylcholine in the medium compared to control fibroblast cultures. Washing labeled lung slices or cells with a nonlysing concentration of Triton X-100 (0.05%) did not selectively release labeled phosphatidylcholine, indicating that any secreted phosphatidylcholine did not adhere to the surface of the lung slices or cells. Experiments were performed to determine whether the small amount of phosphatidylcholine in the medium and detergent-released phosphatidylcholine was similar to the tissue and cell phosphatidylcholine. The saturated fatty acid composition of the phosphatidylcholine released by Triton X-100 and in the medium (from lung slices) was identical to that of the tissue phosphatidylcholine. In addition, the relative labeling rates of the phospholipids released by Triton X-100 and in the medium (labeled with [(14)C]glycerol) were identical to those of the tissue and cell phospholipids. Based on these results, we conclude that phosphatidylcholine is not secreted by lung slices and lung cells in large amounts compared to controls. The implication of these data is that pulmonary surfactant material may actually not be secreted by the lung in vitro, and perhaps in vivo, in the manner that is currently generally accepted.

Effect of hypophysectomy and growth hormone treatment on albumin mRNA levels in the rat liver.

A complementary DNA copy of purified rat albumin mRNA was employed in RNA-excess hybridization reactions to examine albumin mRNA levels in the liver of the hypophysectomized rat. In normal rat liver, albumin mRNA sequences were found to be about 10% of the total poly(A)-containing RNA population. Hypophysectomy reduced albumin mRNA levels by approximately 50%. Growth hormone treatment of hypophysectomized animals returned albumin mRNA to nearly normal levels. These effects on albumin mRNA sequences agreed closely with the changes in the synthesis of albumin that are assoicated with these conditions. The results suggest that hypophysectomy and growth hormone influence albumin synthesis by affecting the level of albumin mRNA.

Histone interactions in solution and susceptibility to denaturation.

Histone interactions in solution may depend upon treatments used for purification. Optical rotatory dispersion and sedimentation-velocity measurements have been made in a reference solvent, before and after exposure to various treatments, to investigate histone susceptibility to irreversible denaturation. Some acid conditions and urea and guanidine solutions may denature. Interaction studies performed on nondenatured histones indicate that the dimer, (H4)(H3), and tetramer, (H4)2(H3)2, dissociate to monomers at low ionic strength. Sedimentation-velocity experiments suggest a model for the (H4)2(H3)2 tetramer, with a compact semispherical center and four protruding amino-terminal regions. Fractions H2a and H2b interact to form the mixed dimer in equilibrium with monomers. Fraction H2a self-associates readily to dimers, tetramers, and octamers, while fraction H1 associates only weakly to form dimers.

Effect of hypophysectomy on the synthesis of rat liver albumin.

Hypophysectomy of adult rats results in approximately a 50% decrease in the rate of albumin synthesis relative to total liver protein synthesis. This decrease is accompanied by a proportional decline in the number of albumin-synthesizing polysomes, as determined by the binding of 125I-Labeled anti-albumin antibody, and indirect immunoprecipitation of [3H]leucine-labeled albumin-synthesizing polysomes. Furthermore, this decrease is associated with an equivalent reduction in the amount of total membrane-bound polysomes, whereas total free polysomes show little quantitative change. The size of the specific albumin-synthesizing polysomes, as well as the size of the total polysomes, however, appear to be the same following hypophysectomy as in the normal untreated animal. These observations are consistent with the finding that the relative amount of albumin mRNA activity also decreases approximately 50%, as assayed in a heterologous cell-free protein-synthesizing system using exogenous liver RNA prepared from either isolated polysomes or total liver homogenates. The decrease in albumin production in the hypophysectomized rat, therefore, is apparently the result of a reduction in the amount of active albumin mRNA. The concomitant decrease in albumin-synthesizing polysomes appears to reflect a similar reduction in the amount of total membrane bound polysomes. Thus, a major physiological defect in hypophysectomy may be a preferential decline in membrane-bount polysomes accompanied by a reduction in mRNA levels, which is represented by the decrease in albumin synthesis.

Bottom currents in the hudson canyon.

In-place measurements of the bottom currents in the Hudson Canyon reveal that the current regime is characterized by a pronounced reversal of flow up and down the canyon. Velocities are commonly of the order of 8 to 15 centimeters per second, reaching 27 centimeters per second on occasion in the upper and central portion of the canyon. Although alpha 2.5-day recording of currents showed a net transport upcanyon, a combination of 66 current measurements from the submersible Alvin, the analysis of sediment texture and organic carbon, and the determination of the benthic fauna-nutrient relationship indicate that over the long term there is a net transport of fine material through the canyon to the outer continenital rise.