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We have purified to near homogeneity the major DNA-dependent ATPase from human cells. The pure enzyme has a mol. wt. of 68,000 and a minimum specific activity of approximately 150 U/mg. When the properties of the pure enzyme are compared with those of a less purified preparation, significant differences are observed both in structure and in function. These can be ascribed to the interaction of the ATPase with a DNA-binding protein (mol. wt. 28,000) that we can also purify to near homogeneity from the same cells and which is present in the less purified preparations of the ATPase. The ability of the less purified ATPase to stimulate DNA polymerase alpha in helicase fashion is probably due to the presence of the DNA-binding protein.

The reproducibility of ambulatory, home, and clinic blood pressures was compared in 13 untreated mildly hypertensive and 14 normotensive subjects. Each subject had two sets of daily ambulatory recordings, home self-measured readings (over 6 days), and clinic measurements taken 2 weeks apart. Comparisons over the 2 weeks within and among the methods of measurements were made using a repeated-measures analysis of variance. The results showed that there was no consistent average change in the ambulatory or home pressures and no change in clinic diastolic pressures, but the clinic systolic pressure of the hypertensive subjects dropped 6 mm Hg (p less than 0.05), while that of the normotensive subjects showed no significant change. Test-retest correlations of each of the three methods were similar in magnitude, indicating a similar level of reliability. Test-retest correlations of the ambulatory standard deviations, however, were low, indicating a low reliability of this measure of daily pressure variability. These results suggest that the reproducibility of ambulatory pressures may be as good or better than that of home or clinic measurements. They also suggest that the average ambulatory pressure may be preferable as the measurement in clinical trials, since it may be less influenced by measurement anxiety, particularly in hypertensive subjects.

A purification procedure for proteins which bind heterogeneous nuclear RNA (hnRNP proteins) is described. The procedure, which entails standard chromatographic fractionations (single-stranded DNA cellulose, hydroxyapatite) and detection with specific antibodies, allows a large-scale preparation of these proteins and the partial separation of different polypeptides. By this method, polypeptides of higher molecular mass (53-55 kDa) can be purified, which are structurally and antigenically related to the 'canonical' hnRNP core proteins (34-43 kDa) that constitute the 40S hnRNP complexes. We also show that HeLa cells contain a protease that cleaves hnRNP core proteins to discrete smaller polypeptides of 22-28 kDa. Such protease, which has been partially purified, appears to copurify extensively with some of the hnRNP proteins.

In this study the specificity of circulating autoantibodies in ANA+ aged donors, ANA- donors and SLE patients was investigated by immunoblotting on total nuclear proteins and by ELISA on purified nuclear proteins, possibly related to DNA metabolism, such as DNA polymerase alpha, DNA-dependent ATPase, DNA Topoisomerase I, ssDBP, hnRNP, HMG and histones. Immunoblotting showed that sera from ANA+ aged donors present fewer antibodies to nuclear proteins, especially to those between 21,000 and 45,000, molecular weight (MW), than sera from SLE patients. When the specificity of antisera was further studied on purified nuclear proteins, it was found that the majority of sera from SLE patients react with most of the proteins tested, whereas sera from ANA+ aged donors mainly react with DNA polymerase alpha, DNA-dependent ATPase, DNA Topoisomerase I and histones. In addition, sera from a few ANA- donors also reacted with certain purified nuclear proteins in a statistically significant age-related manner.

Three DNA-dependent ATPases (gamma phosphohydrolases) can be isolated from Bacillus subtilis cells. We studied these enzymes in a number of mutants deficient in recombination or repair functions (rec, uvr) and in competent cells. The recA mutant studied had lower ATPase II activity, while competent cells had higher ATPase I activity, in comparison with the parental strain not brought to competence.

The hemodynamic effects of combined administration of ibopamine (Ib) (150 mg orally) with nitroprusside (NP) (50-150 micrograms/min intravenously) were compared with those of NP alone in 17 patients with severe congestive heart failure due to coronary artery disease (7 patients) or idiopathic cardiomyopathy (10 patients). Hemodynamic measurements were obtained using a Swan-Ganz thermodilution catheter and a bedside thermodilution cardiac output computer. Nitroprusside alone produced a significant decrease (-12.4%) in mean arterial pressure, mean pulmonary arterial pressure (-28.3%), and systemic vascular resistance (-22.6%), and a significant increase in stroke volume index (23.1%). The administration of combined NP and Ib produced a further significant increase of stroke volume index (20.1%) with a concomitant and significant reduction of systemic vascular resistance (-19.4%); heart rate, mean systemic and pulmonary arterial pressures did not change significantly from the values observed with NP alone. Moreover, stroke work index, although not significantly modified with the vasodilator alone, was significantly increased over control values with NP + Ib association. Although NP alone induced similar effects in both the ischemic and idiopathic cardiomyopathies, the association of Ib gave a more favorable, though not significant, hemodynamic response in the subjects with primitive cardiomyopathy than in the ischemic ones. Thus, the association of Ib to NP therapy, in patients with congestive heart failure, further increases stroke volume index and stroke work index with a concomitant reduction of systemic vascular resistance, without any significant change in mean systemic and pulmonary arterial pressures, or heart rate. These results point out the possibility of associating Ib with other orally active vasodilators in the chronic treatment of congestive heart failure.

Protein factors have been isolated from HeLa cells and from calf thymus which are able to specifically stimulate DNA polymerase alpha in vitro on templates which mimic the replication fork. One factor, extracted from HeLa cells, is an enzymatic complex of about 100-110 Kdal composed of a DNA-dependent ATPase and of an as yet uncharacterized DNA-binding protein. This complex exhibits a limited "helicase" activity on DNA : DNA partial duplexes which probably accounts for the stimulation of DNA polymerase alpha. The other stimulatory factor is obtained from calf thymus. They are the so-called single-stranded DNA binding proteins (DBP) which have a duplex-destabilizing activity. These proteins appear to be heterogeneous with regard to both physical properties (Mr and pI) and functional characteristics (DNA polymerase alpha stimulation, duplex denaturation). The origin and the biological significance of the different molecular forms are discussed.

A comparison of the effects of different factors on the sensitivity of Western blotting technique to detect monoclonal antibodies is described. The major improvements were obtained by: A) renaturating the antigen in the gel before transferring it in carbonate buffer at pH 10 onto nitrocellulose and B) using alkaline-phosphatase-conjugated second antibody instead of peroxidase-conjugated second antibody.

We have isolated a new mutant of Bacillus subtilis temperature sensitive in DNA replication; its properties are those of an initiation mutant. When liquid cultures are shifted to 48 degrees DNA replication is the first macromolecular synthesis that stops, but only after synthesis of the amount of DNA predicted for the completion of one replication round. When spores of the mutant are germinated and shifted to 48 degrees at subsequent times, one round of DNA replication is observed only when the shift occurs between 60 and 100 min; earlier shifts do not allow replication to start, later shifts allow more than one replication. The DNA replicated after a shift to high temperature is enriched in markers close to the terminus. The reinitiation of DNA replication stopped by the high temperature, takes place following a shift to a permissive temperature only if protein synthesis is allowed. Examination of DNA replication following toluene treatment shows that the elongation of DNA chains is not affected at the non-permissive temperature. This mutant is shown by PBS-1 mapping to correspond to a new gene denominated dna P, which is located between the thy A and fur A genes and is distinct from all the mapped dna and rec genes of Bacillus subtilis. The mutation confers to the cells also a deficiency in the ability to be transformed, to be transfected with SPP1 phage DNA, and to survive treatment with methyl-methane sulfonate. These deficiencies, observed at the permissive temperature, are no more temperature dependent than in the parental strain. The ability to perform homologous and heterologous transduction with PBS-1 phage and the sensitivity to ultraviolet radiation or mitomycin C are normal.

The development of bacteriophages SPP1 and phi 29 has been studied in several B. sutilis mutants defective in host DNA replication, under non permissive conditions. Several gene products, involved in the synthesis of host DNA, are required for phi 29 replication, while SPP1 seems to require only the host DNA polymerase III. In addition both phages are unable to grow in a dna A mutant (ribonucleotide reductase). Taking advantage of the fact that SPP1 DNA is actively replicated in several dna mutants at non-permissive temperature, we have studied the structure of the replicative intermediates of this phage in the absence of interfering host DNA synthesis. Fast sedimenting forms of SPP1 DNA can be isolated from phage infected cells and evidence of covalently joined concatemers has been obtained, suggesting the presence of terminally repeated sequences.

The onset of serious arrhythmias during potassium depletion occurs rather frequently in female subjects who have undergone hypotensive-diuretic treatment, independently from the duration and doses of drugs. These arrhythmias which produce a cardiac arrest, can also occur in subjects not affected with heart disease. They are not necessarily preceeded by clinical prodrumus or other types of minor arrhythmias not accompanied by other important electrocardiographic or serum-logical alterations of hypokaliemia. The most commonly observed type is the "torsades de pointe", though cases of ventricular tachycardia or ventricular fibrillation are also documented. The ethiopathogenesis is discussed with regard to the alterations of the basic electrocardiogram as well as to the kind of major arrhythmia. In most cases , lidocaine has given the most satisfactory thmias, results in the treatment of these arrhythmias, probably because of the modality of the action which is substantially different from the other antiarrhythmic drugs.

We tested whether overload of the two ventricles may be associated with a preponderance of interventricular septum in patients with high blood pressure. The rationale is that the septum is shared by the greater and lesser circulation and that in hypertension the latter shows the same qualitative hemodynamic alterations as the former. Among 65 hypertensive patients, 40 (group 1) showed (echo) posterior wall thickness within the mean +/- 1 SD of 62 normal subjects, and 25 (group 2) had a posterior wall thickness exceeding the mean + 1 SD of the normal population. Both groups were subdivided into subgroups A and B, which included patients whose ventricular septum was similar to (within the mean + 1 SD) and thicker than (exceeding the mean + 1 SD) the posterior wall thickness in the corresponding group, respectively. Resting differences in systemic and pulmonary pressure and vascular resistance among subgroups 1A, 1B, and 2A were not significant; however, in subgroup 2B these variables exceeded those in the other subgroups to a significant extent. During cold pressor testing (CPT) the levels reached and the changes in pressure and resistance from baseline values in both circuits were much greater in subgroups B than in subgroups A. The baseline plasma norepinephrine value showed a trend toward an increase from subgroup 1A to 1B and from subgroup 2A and 2B; during CPT norepinephrine invariably changed and in subgroups B it rose significantly more than in subgroups A. It was not determined whether this caused the hemodynamic overload in subgroups B.(ABSTRACT TRUNCATED AT 250 WORDS)

Deoxyribonucleic acid (DNA) extracted from phage SPP1 is highly infectious on Bacillus subtilis competent cells; the efficiency of infection is 5 x 10(3) to 6 x 10(3) phage equivalents per plaque-forming unit. This DNA was used to study the relationship between competence for transfection and for transformation. The experiments were concerned with the frequency of infection and transformation in mutants exhibiting different levels of competence, the effect of periodate on competence for infection and for transformation, the competition between phage and bacterial DNA, the transformation of cells preinfected with phage DNA, and the infection of cells pretreated with bacterial DNA. The data show that B. subtilis cells competent for transformation are also competent for transfection and vice versa; transfection with phage DNA represents, therefore, a simple way to measure the total number of competent cells in a culture. The fraction of competent cells, determined by SPP1 DNA infection, varied from 10(-2) to 7 x 10(-2).

By preventing phage DNA synthesis during a critical period, conditions have been found under which DNA replication and phage production are rescued after infection with T4 DNA ligase mutants.

Subinhibitory doses of rifampin cured F(+)Escherichia coli cells from the episome. The target of the drug was transcription because E. coli mutants with a ribonucleic acid polymerase resistant to rifampin were not cured. The experimental conditions required for optimal curing with rifampin very closely resembled those required for curing with acridine orange. Mutants were found which are more resistant to curing by both acridine orange and rifampin. Probably the two drugs affect a common metabolic step, or alternatively they may inhibit the synthesis of a factor which is necessary for the replication of the episome.