Site-directed psoralen crosslinking of DNA.

A technique has been developed for placing site-specific crosslinks into DNA by using a psoralen derivative containing a sulfhydryl group. Plasmid pBR322 was nicked at the unique BamHI restriction site, and mercurated pyrimidines were introduced by nick-translation. Then the psoralen was specifically directed to this site in the dark through a Hg-S linkage prior to irradiation to form a crosslink. The location of interstrand cross-links was examined by electron microscopy of Pst I-cut molecules and found to be at or near the BamHI site.     

A novel nonphagocytic mechanism of erythrocyte destruction involving direct cell-mediated cytotoxicity

The number of erythrocytes fell when co-cultured with cell preparations derived from mouse spleen, thymus, bone marrow, or peritoneal exudate (PE) cells. Erythrocyte-depletion activities (EDA) of different leukocyte preparations were in the order PE > spleen > thymus > bone marrow. Adherent, nonadherent, T-depleted, and T-enriched cell subpopulations had comparable EDA. Spleen cells from athymic nude mice, however, lacked significant EDA. In addition, EDA was boosted by Concanavalin A (Con A) but not by lipopolysaccharide, indicating that T cells may play a crucial role in inducing EDA in spleen cells. Paraformaldehyde-fixed spleen or PE cells, as well as membrane preparations isolated from spleen cells, efficiently lysed erythrocytes. Erythrocyte ghost membranes inhibited erythrocyte lysis by control or paraformaldehyde-fixed spleen cells. Treatment with hamster anti-mouse Fas or anti-mouse tumor necrosis factor receptor (TNFR) antibody could opsonize erythrocytes for faster depletion by spleen cells, suggesting an expression of Fas and TNFR on erythrocytes. TNF alpha could lyse erythrocytes in a dose-dependent fashion. Additionally, enhanced spleen cell EDA induced in response to succenyl Con-A could be blocked by anti-TNF alpha antibodies. Our results provide evidence for a direct cell-mediated cytotoxicity (CMC) of erythrocytes by leukocytes. A role of molecules of Fas and the TNF family in CMC of erythrocytes has also been suggested. Further work is needed to understand if, and to what extent, CMC of erythrocytes contributes to erythrocyte destruction in vivo and to determine its patho-physiological significance.     

Glucocorticoid-thyroid synergism in lung maturation: a mechanism involving epithelial-mesenchymal interaction.

We studied the effects of cortisol and triiodothyronine (T3) on 20-day fetal rat lung cell cultures. Cortisol enhanced the production of surfactant-associated saturated phosphatidylcholine while T3 did not. However, T3 potentiated the cortisol effect. We observed that T3 enhanced the response of cultures enriched with alveolar type H cells to fibroblast-pneumonocyte factor (FPF). Intracellular cAMP was increased by exposure of these cultures to FPF, and T3 potentiated this increase. Unlike cortisol, T3 had no effect on production of FPF by fetal lung fibroblasts, as determined by bioassay of fractions of fibroblast-conditioned medium partially purified by column chromatography. The time course of cortisol action on mixed (fibroblast/epithelial) cultures was in keeping with the proposed mechanism: glucocorticoid induction of FPF in fibroblasts, followed by FPF induction of cAMP in epithelia and, finally, by enhanced production of saturated phosphatidylcholine. Thus, glucocorticoid acting on mesenchyme and thyroid hormone acting on epithelium have a synergistic effect on expression of differentiated epithelial function.     

Estrogen-induced endogenous DNA adduction: possible mechanism of hormonal cancer.

In animals and humans, estrogens are able to induce cancer in susceptible target organs, but the mechanism(s) of estrogen-induced carcinogenesis has not been elucidated. A well-known animal model is the development of renal carcinoma in estrogen-treated Syrian hamsters. Previous work demonstrated the presence of covalent DNA addition products (adducts) in premalignant kidneys of hamsters exposed to the synthetic estrogen, diethylstilbestrol, a known human carcinogen. In the present study, the natural hormone, 17 beta-estradiol, and several synthetic steroid and stilbene estrogens were examined by a 32P-postlabeling assay for their capacity to cause covalent DNA alterations in hamster kidney. Chronic exposure to each of the estrogens tested led to the gradual formation of five chromatographically distinct unusual nucleotides specifically in kidney DNA. Irrespective of the estrogen used, chromatograms exhibited identical mobilities of each of these adducts in seven different systems on PEI-cellulose anion-exchange TLC, in three different conditions on reversed-phase TLC, and in one system on silica gel partition TLC. Therefore, the DNA adducts observed did not contain moieties derived from the structurally diverse estrogens. It is concluded that each of the estrogens induced the binding of the same unknown endogenous compound (or compounds) to target tissue DNA. This novel property of estrogens is postulated to play a key role in hormone-induced malignancy.     

Site-specific interaction of DNA gyrase with DNA.

DNA gyrase, in the presence of the inhibitor oxolinic acid, can induce double-strand DNA breakage at specific sites. The sequences at several sites have been determined. In addition, the structure of complexes formed between DNA gyrase and restriction fragments containing an oxolinic acid-promoted cleavage site has been examined by DNase protection methods. DNA gyrase protects more than 120 base pairs of DNA against pancreatic DNase in a region surrounding the cleavage site. Protection is observed both in the presence and absence of oxolinic acid. Protected DNA flanking the cleavage site contains DNase I-sensitive sites spaced on the average 10 or 11 base pairs apart. This result supports the view that, in the DNA gyrase--DNA complex, the DNA is largely wrapped on the outside of the enzyme.     

A mechanism of DNA transposition.

Bacteriophage Mu and many other transposable elements undergo transposition by a process that involves replication of the element. We describe here a mechanism by which such integrative replication may take place. We hve examined electron microscopically the DNA structures generated in host cells after Mu induction and have deduced the following steps in the transposition process, (i) Association. A protein-mediated association is brought about between the transposable element and the target DNA. (ii) Attachment. One end of the element is nicked and attached to a site that undergoes a double-stranded cleavage. (iii) Roll-in replication. While one strand of the target DNA is linked to the nicked strand of the element, the complementary strand of the target DNA is used as a primer for replication into the element such that the replicating DNA is threaded through the replication complex. (iv) Roll-in termination. When the distal end of the element arrives at the replication complex, replication is terminated. The roll-in replication mechanism can also explain laying down of tandem repeats--i.e., amplification of circular DNA sequences.     

A possible mechanism for pacemaker-induced T-wave changes.

The genesis and the significance of pacemaker-induced T-wave changes remain unclear. Changes in body surface potential mapping (BSM) were observed and compared with resting thallium-201 myocardial scintigraphy (T1-SC) findings before, during and after ventricular pacing (VP) in 10 patients with various bradyarrhythmias. All studies were performed with the patients taking no medication. In all patients, isoarea QRST maps showed a characteristic abnormal dipolar pattern with positive values distributed over the upper chest and negative values over the lower chest during VP at a physiological rate for 14 days or more. These abnormalities were preserved almost completely after pacing was terminated; and coincided with deep T-wave inversions in leads II, III, aVF and V4-6. In three patients, BSM performed before VP showed normal QRST isoarea maps with positive values distributed over the left lower chest. All patients in whom resting T1-SC was performed during chronic VP showed transient perfusion defects in the posteroinferior (seven cases) or inferolateral (one case) left ventricular wall. In three patients, T1-SC was performed before VP and showed a normal distribution. Both the pacing-induced perfusion defects and the T-wave abnormalities remained unchanged 2 h after ceasing VP, were attenuated 7 days later and disappeared within a month. These findings suggest that chronic ventricular pacing may produce myocardial ischaemia, and that it persists for a certain period after the cessation of pacing, resulting in post-pacing T-wave inversion.     

Shoshin beriberi with vasospastic angina pectoris possible mechanism of mid-ventricular obstruction: possible mechanism of mid-ventricular obstruction.

A 73-year-old heavy drinker was admitted to hospital in a state of shock. He had been suffering from frequent angina at rest, causing him to drink more heavily in an effort to overcome his anginal chest pain. He had been drinking hard each day and had not eaten for 4 weeks. His hemodynamic state on admission showed high-output heart failure. Echocardiography revealed hyperkinesis of the left ventricle and mid-ventricular obstruction with peak intraventricular gradients of 30 mmHg. Although no improvement was seen despite administering the maximal dose in catecholamine therapy, his condition improved rapidly after vitamin B(1) was administered. Cardiac catheterization revealed mid-ventricular obstruction with an apical aneurysm. Coronary artery spasm was induced by injecting acetylcholine in the distal site of the left anterior descending artery, which perfused the area of the apical aneurysm. In the present case, both left ventricular hyperkinesis caused by shoshin beriberi and apical myocardial infarction caused by frequent coronary spasms produced mid-ventricular obstruction with an apical aneurysm.     

Targeted mutagenesis of DNA using triple helix-forming oligonucleotides linked to psoralen.

Oligonucleotides can bind as third strands of DNA in a sequence-specific manner in the major groove in homopurine/homopyrimidine stretches in duplex DNA. Here we use a 10-base triplex-forming oligonucleotide linked to a psoralen derivative at its 5' end to achieve site-specific, targeted mutagenesis in an intact, double-stranded lambda phage genome. Site-specific triplex formation delivers the psoralen to the targeted site in the lambda DNA, and photoactivation of the psoralen produces adducts and thereby mutations at that site. Mutations in the targeted gene were at least 100-fold more frequent than those in a nontargeted gene, and sequence analysis of mutations in the targeted gene showed that 96% were in the targeted region and 56% were found to be the same T.A to A.T transversion precisely at the targeted base pair. The ability to reproducibly and predictably target mutations to sites in intact duplex DNA by using modified oligonucleotides may prove useful as a technique for gene therapy, as an approach to antiviral therapeutics, and as a tool for genetic engineering.     

Termination sites of the in vitro nick-translation reaction on DNA that had photoreacted with psoralen.

A double-stranded circular DNA having a single nick at a specific site has been photochemically induced to react with 4'-hydroxymethyl-4,5', 8-trimethylpsoralen (HMT) and used as a substrate for nick-translation with Escherichia coli DNA polymerase I holoenzyme. By using the dideoxy chain-terminating sequencing procedure, it was possible to map the termination sites observed on the template that had photoreacted with HMT. These sites occur at nucleotides preceding potential psoralen crosslinking sites. Analysis of DNA products synthesized on templates that had photoreacted under conditions designed to maximize psoralen monoaddition revealed that DNA polymerase I is not stopped by this lesion. Psoralen monoadducts situated on the template strand act only as kinetic attenuators, whereas psoralen monoadducts localized on the nick-translated strand have no effect on the rate of synthesis. These data suggest that psoralen crosslinks are responsible for the lethal effects of psoralen photochemistry in E. coli. Mutagenesis may be associated, however, with the repair replication of psoralen monoadducts by E. coli DNA polymerase I.     

Induction and the possible mechanism of ventricular tachycardia after catheter ablation with direct current shocks in dogs

Cathodal DC shocks (150-J) were administered via the His bundleto 20 closed-chest dogs, and in a further three dogs 25-J cathodalshocks were given via the left ventricular endocardium. In 18dogs, including three that underwent left ventricular ablation,Holter electrocardiograms were recorded from 1 to 7 days afterablation, and 4 weeks after ablation. There were frequent episodesof sustained ventricular tachycardia (VT) from the first fewhours after ablation to 4 days after ablation in all dogs, butboth the rate and the coupling interval of VT were variable.In five conscious dogs stimulated 1 day after ablation, it wasdifficult to induce and terminate VT repeatedly. There was adirect relationship between the paced cycle length and the intervalof the last paced beat to the initiating VT beat in three outof four dogs. In the fourth dog there was an inverse relationship.There was no transient entrainment with ventricular burst pacingduring VT in any of the four dogs tested. The effects of lidocaine(2–3 mg.kg^–1), verapamil (0.2–0.4 mg.kg^–1),and propranolol (0.2 mg.kg^–1) on VT were tested within2 days of ablation in 10 conscious dogs. In general, both lidocaineand verapamil terminated VT, and propranolol slowed VT. In conclusion,VT soon after ablation possibly results from triggered activity,although abnormal automaticity cannot be ruled out.     

Adenovirus recruits dynein by an evolutionary novel mechanism involving direct binding to pH-primed hexon

Following receptor-mediated uptake into endocytic vesicles and escape from the endosome, adenovirus is transported by cytoplasmic dynein along microtubules to the perinuclear region of the cell. How motor proteins are recruited to viruses for their own use has begun to be investigated only recently. We review here the evidence for a role for dynein and other motor proteins in adenovirus infectivity. We also discuss the implications of recent studies on the mechanism of dynein recruitment to adenovirus for understanding the relationship between pathogenic and physiological cargo recruitment and for the evolutionary origins of dynein-mediated adenovirus transport.