Fate of transforming bacteriophage HP1 deoxyribonucleic acid in Haemophilus influenzae lysogens.
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The biological fate of temperate phage HP1 deoxyribonucleic acid (DNA) was followed after uptake by defectively lysogenic competent Haemophilus influenzae cultures. The similar inactivation kinetics of three single phage genetic markers and of their triple combination indicated a complete rather than partial destruction of about half of the adsorbed DNA molecules. Intracellular DNA breakdown products were tentatively identified by hydroxyapatite column chromatography as short single strands and extensively damaged short double strands. Integrated donor DNA (after single-strand insertion?) was still highly efficient for triple-marker co-transformation. This suggests that whole or nearly whole donor DNA molecules were integrated. Some donor DNA was never integrated but remained largely unaltered. This DNA fraction did not contain significant amounts of recipient prophage marker activity. It is concluded that it had not participated in some kind of reciprocal recombination event involving the recipient chromosome. Since very similar phage DNA marker inactivation rates were observed after adsorption by competent nonlysogenic recipients (transfection), the relationship between biological inactivation of adsorbed donor phage DNA and its integration in lysogenic recipients is not clear. (+info)
Concentrations of tamoxifen and its major metabolites in hormone responsive and resistant breast tumours.
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Patients treated with tamoxifen (TAM) for primary breast cancer often manifest de novo or acquired resistance, possibly through changes in drug metabolism. Using solid-phase extraction methods and reversed-phase high-performance liquid chromatography separations, levels of TAM and metabolites 4-hydroxytamoxifen (4OH) and desmethyltamoxifen (DMT) have been measured in plasma and tumour tissue from breast cancer patients treated with TAM for at least 3 months. Patients were categorized into those with tumours responding to TAM and those showing de novo or acquired resistance. Levels of TAM, 4OH and DMT in both plasma and tissue samples were correlated with clinical response, length of treatment and patient weight. Interesting results included accumulation of 4OH in tumour tissues over time in all patients, with significance reached in the acquired resistance group. In addition, significantly lower levels of 4OH and DMT were found in plasma taken from responding patients after 3 months of treatment when compared to non-responding patients, and a small group of ER-poor patients showed significantly lower levels of all three species in plasma when compared to other patients. Whilst not explaining TAM resistance in all cases, these differences could account for the development of resistance to TAM treatment in certain subgroups of patients. (+info)
Terminal deoxynucleotidyl transferase activities in human blood leukocytes and lymphoblast cell lines: high levels in lymphoblast cell lines and in blast cells of some patients with chronic myelogenous leukemia in acute phase.
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Terminal deoxynucleotidyl transferase, an enzyme which catalyzes the polymerization of deoxyribonucleoside triphosphates, elongating oligo- or polydeoxynucleotide chains, but without direction from a nucleic acid template, is thought to be specific for thymus gland and thymus-derived cells. We have confirmed the observations that high levels are characteristic of thymus gland with both human and calf tissue and that elevated levels may be found in some cases of acute lymphocytic leukemia. High levels were also found in human lymphoblast cell lines with T-cell characteristics, and insignificant activity was observed in leukocytes of patients with chronic myelogenous leukemia not in acute blast phase of the disease, chronic lymphocytic leukemia, human B-cells, and normal human blood lymphocytes even after stimulation with phytohemagglutinin. However, high levels (approximately 200 nmoles/hr/10(9) cells) equivalent to those in thymus tissue and lymphoblast cell lines with T-cell characteristics were found in the peripheral blood blast cells of four patients with chronic myelogenous leukemia in an acute blast phase of their disease. One hypothesis that may explain the present results is that in chronic myelogenous leukemia in acute blast phase of the disease the proliferative blast response may not always be myeloblasts but in some cases it may be lymphoblasts. (+info)
Deoxynucleotide-polymerizing enzyme activities in T- and B-cells of acute lymphoblastic leukemia origin.
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All 5 thymus-dependent cell (T-cell) lines (Molt-3; Molt-4; RPMI-8402; CCRF-CEM; CCRF-HSB-2) and 7 thymus-independent cell (B-cell) lines (RPMI-8382, RPMI-8392, RPMI-8412, RPMI-8422, RPMI-8432, RPMI-8442, CCRF-SB) established so far from acute lymphoblastic leukemia patients were examined for deoxynucleotide polymerizing enzymes. All T- and B-cells had DNA polymerase gamma, DNA polymerase beta, and terminal deoxynucleotidyl transferase both in the soluble (the latter 2 enzymes only in small amounts) and chromatin fraction, whereas DNA polymerase alpha was found only in the soluble fraction. With respect to their sedimentation and chromatographic behavior, template-primer requirements, Km for deoxythymidine triphosphate or deoxyguanosine triphosphate divalent cation preference, effect of NaCI and inhibitors, the enzymes from T- and B-cells resembled each other and those from other mammalian cells. DNA polymerase alpha, beta, and gamma from T-cells like those from "fresh" acute lymphoblastic leukemia cells, were more thermolabile than those from B-cells or phytohemagglutinin-stimulated normal lymphocytes. In addition, the terminal deoxynucleotidyl transferase from the above cells was completely inactivated in 5 to 6 min at 50 degrees, whereas the DNA polymerase alpha, beta, and gamma retained considerable activity even after heating for 25 min at 50 degrees. DNA polymerase activity of the soluble fraction from T-cells was of the same magnitude as in B-cells when expressed on a DNA basis but twice that of B-cells when expressed on a protein basis. High terminal deoxynucleotidyl transferase activity, equivalent to that observed in acute lymphoblastic leukemia cells, was found in all T-cell lines that, when expressed on a DNA basis, was 30 to 100 times higher than the B-cell lines tested. These results support the suggestion of earlier investigators that T-cell lines examined here may have originated from leukemic cells. (+info)
Characterization of an 84 kDa protein inducing an immediate hypersensitivity reaction in rabbits sensitized to Haemaphysalis longicornis ticks.
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An immunogenic 84 kDa protein was isolated and purified from whole tick extracts of Haemaphysalis longicornis larvae by a combination of ion exchange, reverse phase and hydrophobic interaction chromatographies. The protein, when injected intradermally into rabbits exposed to repeated tick feeding, induces an immediate cutaneous hypersensitivity reaction. It has been purified to homogeneity as shown by sodium dodecyl sulphate polyacrylamide gel electrophoresis and silver staining. Amino acid sequences for two peptides derived from proteolytic cleavage of p84 were scanned against known proteins on the SWISS-PROT database. A 7 residue motif, ISGWGNT present in one of the two peptides appeared conserved in both vertebrate and invertebrate trypsin-like serine proteinases, while another 7 amino acid motif, HVPAGQI present in the second peptide showed homology to an Escherichia coli ATP-binding protein. We have discussed our findings in relation to isolation and characterization of target antigens for tick vaccine candidates. (+info)
Radioligand receptor assay for 25-hydroxyvitamin D2/D3 and 1 alpha, 25-dihydroxyvitamin D2/D3.
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A competitive protein binding assay for measurement of the plasma concentration of 1 alpha, 25-dihydroxyvitamin D3 [1alpha, 25-(OH)2D3] has been extended to include the immediate precursor of this hormone, 25-hydroxyvitamin D3 (25-OHD3). In addition, the assay system is capable of measuring the two metabolic products of ergocalciferol, namely. 25-hydroxyvitamin D2 (25-OHD2) and 1alpha, 25-dihydroxyvitamin D2 [1alpha, 25-(OH)2D2]. The target tissue assay system consists of a high affinity cytosol receptor protein that binds the vitamin D metabolites and a limited number of acceptor sites on the nuclear chromatin. By utilizing a series of chromatographic purification steps, a single plasma sample can be assayed for any of the four vitamin D metabolites either individually or combined. Therefore, the assay procedure allows for both the quantitative and qualitative assessment of the total active vitamin D level in a given plasma sample. To show that the binding assay was capable of measuring 1alpha, 25-(OH)2D2 as well as 1alpha, 25 (OH)2D3, two groups of rats were raised. One group, supplemented with vitamin D3, produced assayable material that represented 1alpha, 25-(OH)2D3. The other group, fed only vitamin D2 in the diet, yielded plasma containing only 1alpha, 25-(OH)2D2 as the hormonal form of the vitamin. The circulating concentrations of the two active sterols were nearly identical (15 ng/100 ml) in both groups, indicating that the competitive binding assay can be used to measure both hormonal forms in plasma. In a separate experiment, 1alpha, 25-(OH)2D2 was generated in an in vitro kidney homogenate system using 25-OHD2 as substrate. Comparison of this sterol with 1alpha, 25-(OH)2D3 in the assay system showed very similar binding curves; the D2 form was slightly less efficient (77%). Comparison of the respective 25-hydroxy forms (25-OHD2 vs. 25-OHD3) at concentrations 500-fold that of 1alpha, 25-(OH)2D3, again suggested that the binding of the D2 metabolite was slightly less efficient (71%). Finally, the assay was employed to measure the total active vitamin D metabolite pools in the plasma of normal subjects and patients with varying degrees of hypervitaminosis D. The normal plasma levels of 25-OHD and 1alpha, 25-(OH)2D measured in Tucson adults were 25-40 ng/ml and 2.1-4.5 ng/100 ml, respectively. Both sterols were predominately (greater than 90%) in the form of vitamin D3 metabolites in this environment. Typical cases of hypervitaminosis D exhibited approximately a 15-fold increase in the plasma 25-OHD concentration, and a dramatic changeover to virtually all metabolites existing in the form of D2 vitamins. In contrast, the circulating concentration of 1alpha, 25-(OH)2D was not substantially enhanced in vitamin D-intoxicated patients. We therefore conclude that hypervitaminosis D is not a result of abnormal plasma levels of 1alpha, 25-(OH)2D but may be cuased by an excessive circulating concentration of 25-OHD. (+info)
Concentration and purification of enteroviruses by membrane chromatography.
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A simple procedure for the concentration and partial purification of enteroviruses from tissue culture harvests is described. After removal of acid-precipitating components with a cationic detergent, the detergent and most membrane-coating components were removed by treatment with a cationic-exchange resin. The resin effluent was then acidified, and the virus was adsorbed to epoxy-fiberglass membranes. Virus was then eluted with pH 11.5 glycine-NaOH buffer. Since this eluate contains no orgcentrated simply by acidifying the eluate and passing it through a smaller membrane than that used for the first concentration. As high as 500-fold concentrations can be achieved, with a high efficiency of recovery. (+info)
Studies on human antihemophilic factor. Evidence for a covalently linked subunit structure.
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When purified antihemophilic factor (Factor VIII) was rechromatographed on 4% agarose in 0.15 M NaCl or 1.0 M NaCl, a single protein peak, containing both procoagulant activity and von Willebrand factor activity, as defined by ristocetin-induced platelet aggregation, was eluted in the void volume. Purified Factor VIII immediately lost about 30% of its procoagulant activity when dissolved in 0.25 M CaCl2, and when rechromatographed on 4% agarose in 0.25 M CaCl2, the protein peak and von Willebrand factor activity remained coincident in the void volume; however, most of the remaining procoagulant activity was eluted after the void volume. The elution position of Factor VIII procoagulant activity from 4% agarose in 0.25 M CaCl2, and hence its apparent molecular weight, varied with the protein concentration applied to the column; at low protein concentrations it was eluted close to the inner volume. Yet on Sephadex G-200 in 0.25 M CaCl2, the protein and procoagulant activity were eluted together in the void volume. These observations suggested that the Factor VIII procoagulant activity was not eluting according to size or shape, but was adsorbing to some extent to the agarose. Isolated activity peak material from the 0.25 M CaCl2 columns contained protein and had a typical ultraviolet spectrum. Even at high concentrations, the protein contained no thrombin, Factors IX, X, or Xa activity, or detectable phospholipid. In addition to Factor VIII procoagulant activity, which could be inactivated by a human antibody to Factor VIII, the activity peak protein also contained von Willebrand factor activity. Like native Factor VIII and the void volume protein, the activity peak contained protein that did not enter a sodium dodecyl sulfate 5% polyacrylamide gel in the absence of reducing reagent. After reduction of disulfide bonds, several subunits ranging from 195,000 to 30,000 daltons were observed. These results indicate that the protein in the shifted Factor VIII procoagulant activity peak is large and that its anomalous elution pattern from 4% agarose in 0.25 M CaCl2 results from interaction with the agarose. The Factor VIII-like properties of the activity peak protein and its electrophoretic pattern on sodium dodecyl sulfate gels suggest that it is a species of Factor VIII modified by proteolytic cleavage. These results allow an interpretation that is different from the recently proposed "carrier protein-small active subunit" hypotheses for the structure-function relationships of the Factor VIII molecule. (+info)