Purification of small polydisperse circular DNA of eukaryotic cells by use of ATP-dependent deoxyribonuclease.

Small polydisperse circular (spc) DNAs of mouse thymocytes were purified by a procedure involving nitrocellulose column chromatography and the treatment of ATP-dependent DNase, which acts only upon linear DNA molecules. Nitrocellulose column chromatography prior to the enzyme treatment was essential because digestion of linear DNA duplexes by the enzyme was inhibited by the presence of concomitant single-stranded DNAs. Mitochondrial DNAs were eliminated by linearization with XhoI and digestion with ATP-dependent DNase. The size distribution of the purified spc DNA molecules ranged from 0.2 micron to more than 28 micron, with a mean length of 5.4 micron. Circular molecules of more than 0.4 micron long (or 1.2 kb) were free from the contamination of linear DNA fragments and pure enough to be cloned into plasmids.

Purification and properties of adenosine 5'-triphosphate-dependent deoxyribonuclease from Thermus thermophilus HB8.

An ATP-dependent DNase has been purified from Thermus thermophilus HB8 by a procedure involving streptomycin precipitation, DEAE-cellulose chromatography, Sephadex G-200 gel filtration and heparin-agarose affinity chromatography. ATP-dependent DNase activity was separated into two distinct peaks, Peak A and Peak B, by heparin-agarose affinity chromatography. Each peak fraction was further purified by ATP-agarose affinity chromatography. Peak A and Peak B were eluted from an ATP-agarose column at 0.14 M and 0.28 M KCl, respectively, each as a single peak. Both enzyme activities require ATP and Mg2+ for the degradation of double- and single-stranded DNAs, and degrade denatured DNA about 1.5 times faster than native DNA. The two peaks are optimally active at 69 degrees C and have similar optimal pH ranges from 8.2 to 9.2. The two purified peaks were unstable on storage at -20 degrees C, but were remarkably stabilized by addition of 0.4 mg/ml bovine serum albumin. Ammonium sulfate strongly inhibits the activities of both peaks. The molecular weights of Peak A and Peak B are about 170,000 as estimated by glycerol gradient sedimentation. The average chain lengths of denatured DNA produced by Peak A and Peak B were 4.2 and 3.6, respectively, and the products were terminated by 5'-phosphoryl and 3'-hydroxyl groups. The limit-digested products of denatured DNA produced by Peak B consist of mono-, di-, tri-, tetra-, and pentanucleotides along with some larger fragments. The mode of action of both activities is processive and Peak A does not attack double-stranded circular DNA.

Purification and subunit structure of recBC DNase from Escherichia coli harboring a recB and recC genes-inserted plasmid.

recBC DNase of Escherichia coli has been purified from the transformant, HB101/pFS11-04 (recB+ recC+), by successive ammonium sulfate fractionation, DEAE-cellulose chromatography, Sephadex G-150 gel filtration, hydroxyapatite chromatography, DNA cellulose affinity chromatography, and second DEAE-cellulose chromatography. The purified enzyme was obtained in an overall yield of 3%. The enzyme protein appeared as a single pure component on native polyacrylamide gel electrophoresis. The purified enzyme was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional electrophoresis. The results show that recBC DNase consists of two nonidentical subunits with molecular weights of 125,000 and 135,000, and isoelectric points of 5.6 and 5.7, respectively.

Purification and properties of an acid deoxyribonuclease from rat small intestinal mucosa.

An acid deoxyribonuclease has been purified from rat small intestinal mucosa by a procedure including ammonium sulfate fractionation, chromatographies on DEAE-cellulose, CM-cellulose and SE-Sephadex and finally isoelectric focusing. Polyacrylamide gel electrophoresis of the purified enzyme preparation showed one major and two minor bands, and the enzyme activity corresponded to one of the minor bands. The enzyme preparation was free of contaminating DNase I, DNase III, alkaline RNase, acid and alkaline phosphatases and nonspecific phosphodiesterase, but slight activities of DNase IV and acid RNase were detected. The enzyme did not require divalent cations for activity, had a pH optimum of 4.5 in 0.33 M sodium acetate buffer, and had an optimum temperature of 50 to 60 degrees C when assayed for 30 min. The rate of hydrolysis of native DNA was about 2.5-fold faster than that observed with denatured DNA. Its molecular weight was found to be 9.0 +/- 0.1. The enzyme catalyzes the endonucleolytic cleavage of native and denatured DNA, yielding oligonucleotides which have an average chain length of about 7, and which contain 3'-phosphoryl termini. The mode of action of the enzyme is double-strand scission.

Paralytic ileus due to strongyloidiasis: case report and review of the literature.

We report a rare case of a patient with ileus due to Strongyloides infection that occurred four times within a six-month period. The ileus was improved by treatment with ivermectin and there has not been a recurrence of the symptoms within the last two years.

Thermal stability of nanocrystalline diamond films grown by biased enhanced microwave plasma chemical vapor deposition.

The thermal stability of nanocrystalline diamond (NCD) films grown on mirror-polished silicon substrates by biased enhanced microwave plasma chemical vapor deposition was investigated. Different pieces of a NCD sample were annealed for 1 h in an ambient argon atmosphere at 200, 400, 600, and 800 degrees C. The structural and mechanical properties of as-grown and annealed samples were assessed. The surface roughness and high hardness of the samples remained fairly constant with annealing temperature.

Analysis of serum elements and the contaminations from devices used for serum preparation by inductively coupled plasma mass spectrometry.

Two different samples of serum were prepared from a blood specimen by using two types of serum-separation tubes. Each serum sample was diluted by 100 times with deionized and sub-boiling distilled water. Thirty-six elements in the serum and exudate, from devices used for the serum preparation, were determined by inductively coupled plasma mass spectrometry (ICP-MS). From the comparison of elemental concentrations in two groups of 11 serum samples, it was found that the exudate from serum-separation tubes, as well as from disposable stainless steel needles, had serious effects on the elemental concentrations in the serum. Means for the concentrations of Li, Co, Ga, Cd, Sn, Sb, Ba, La, and Ce in the two serum groups were statistically different from each other. The concentrations of Cr, Mn, Fe, and Mo in both groups were apparently higher than those reported in the literature, suggesting contamination from the disposable stainless steel needle and spectral interferences due to molecular species produced in the argon plasma.

[Clinical efficacy of sulbactam/cefoperazone for the treatment of geriatric patients with respiratory tract infections].

We performed clinical studies on sulbactam/cefoperazone (SBT/CPZ) for the treatment of geriatric patients with respiratory tract infections. Seven patients with pneumonia, 7 with acute bronchitis, 6 with chronic respiratory tract infections were treated with SBT/CPZ. The patients were administered with a daily dose of 2.0 g or 4.0 g for 4-14 days. The clinical responses were excellent in 3, good in 13, fair in 3, and poor in 1 patients. The efficacy rate was 80.0%. No side effects were observed in any patients, but elevations of GOT, GPT were observed in two cases. Causative organisms were E. coli (2 strains), P. aeruginosa (2), MSSA (1), MRSA (1), S. pneumoniae (1), H. influenzae (1), K. oxytoca (1), and E. aerogenes (1). The bacteriological effect rate was 60%. One strain of MRSA and one of two strains of P. aeruginosa persisted in 2 patients.

[A case of myelofibrosis that developed polycythemia vera following treatment with ranimustine and then acute myelogenous leukemia (M0)].

A 60-year-old Japanese woman was admitted to our hospital because of fatigue, weight loss and abdominal distension. Myelofibrosis was diagnosed, based on anemia, huge hepatosplenomegaly, leukoerythroblastosis and bone marrow fibrosis. Following treatment with ranimustine, anemia and splenomegaly improved. Seven months after initial therapy of ranimustine, however, polycythemia (RBC 7.39 x 10(6)/microliter; Hb 19.1 g/dl, Ht 65.9%) developed gradually, then RBC decreased to normal level following venesection (total 1,200 ml). After 32 months, blastic transformation occurred. The blasts were negative for myeloperoxidase. By flow cytometric analysis, the cells were positive for CD2, CD13, CD33 and HLA DR. Thus, AML (M0) was diagnosed. Despite of treatment with multicytotoxic agents, she died of DIC 36 months after the initial diagnosis of myelofibrosis. The progression from myelofibrosis to polycythemia is rare and only 15 cases have been reported so far. In addition, although a chromosomal abnormality, 46, XX, t(3; 12) (q25; p11), was present at the time of first diagnosis of myelofibrosis, the development of an additional abnormality, del(11) (q-), might be related to the transformation to AML.

[Anti-platelet antibody and severe thrombocytopenia during interferon-alpha therapy for chronic active hepatitis C].

We herein report a case of chronic hepatitis C where the patient developed severe thrombocytopenia during interferon therapy. The patient was a 61-year-old woman, who received interferon therapy on April 27, 1993 under the diagnosis of C type chronic active hepatitis. After 4 weeks, her platelet count had decreased to 18,000/microliters and intraoral hemorrhage had begun. Although she received 250 mg of methylprednisolone and 20 U of platelet transfusion three times, her platelet count continued to decrease to 4,000/microliters on both May 28, and on June 3, 1993, and so she was transferred to our hospital on June 4. On her second admission to our hospital, although the platelet-associated IgG (PA-IgG) had increased markedly and the megakaryocytes in her bone marrow had decreased, her platelet count had already increased to 37,000/ microliters, and this gradually returned to a normal level accompanied with a decrease of PA-IgG within one month In this case, although we found immunological abnormalities (high level of IgG, positive ANA and positive anti-smooth muscle antibody) prior to interferon treatment, we could not diagnose the patient as having suffered from autoimmune disease, including autoimmune hepatitis, because she did not satisfy the necessary criteria and because she did not have any symptoms suggesting autoimmune disease. We consider that there may be the possibility that interferon induced only an anti-platelet antibodies that caused the high level of PA-IgG and decreased the production level of platelets within the bone marrow.

[A case of lymphocytic interstitial pneumonia].

A 56-year-old female was admitted on November 1995 to our hospital because of the abnormal shadow on her chest X-ray. Although the chest X ray film revealed diffuse reticulonodular shadows in the bilateral lung fields and right hilar lymphadenopathy, she had not any complaints. Furthermore, mediastinal lymphadenopathy and polyclonal hypergammaglobulinemia were noted. For a further examination, transcutaneous thoracoscopic lung biopsy was performed on August 1996. The lung specimens showed a interstitial infiltration of small lymphocytes exclusively around bronchioles. And the diagnosis of lymphocytic interstitial pneumonia (LIP) was made. She had been suffered from bronchial asthma for 27 years. This is the first report of LIP accompanied with bronchial asthma. Its relationship between LIP and bronchial asthma remains unclear. In the 2 years of follow-up, she remained asymptomatic with unchanged chest radiogram. And her pulmonary function was preserved for the 2 years. But lymphocytic interstitial pneumonia may induce malignant lymphoproliferative disease potentially, we should carefully follow up.

[A case of SLE with the onset of pleuritis showing eosinophilia and elevation of serum IgE].

A 19 year old female was admitted to our hospital with complaints of fever, dyspnea and chest pain. Chest x-ray film showed a massive right pleural effusion. She was diagnosed to have systemic lupus erythematosus (SLE) because of malar rash, serositis (pleuritis), positive antinuclear antibody and positive anti-DNA antibody. Then she was successfully treated with 50 mg/d prednisolone. This case was unusual and of interest in that she had eosinophilia in the peripheral blood and exudative pleural effusion and a marked elevation of serum IgE level despite no history of allergic diseases and no evidence of parasite infections.

Spatial processing in the monkey frontal eye field. I. Predictive visual responses.

Neurons in the lateral intraparietal area and intermediate layers of the superior colliculus show predictive visual responses. They respond before an impending saccade to a stimulus that will be brought into their receptive field by that saccade. In these experiments we sought to establish whether the monkey frontal eye field had a similar predictive response. We recorded from 100 presaccadic frontal eye field neurons (32 visual cells, 48 visuomovement cells, and 20 movement cells) with the use of the classification criteria of Bruce and Goldberg. We studied each cell in a continuous stimulus task, where the monkey made a saccade that brought a recently appearing stimulus into its receptive field. The latency of response in the continuous stimulus task varied from 52 ms before the saccade to 272 ms after the saccade. We classified cells as having predictive visual responses if their latency in the continuous stimulus task was less than the latency of their visual ON response to a stimulus in their receptive or movement field as described in a visual fixation task. Thirty-four percent (11 of 32) of the visual cells, 31% (15 of 48) of the visuomovement cells, and no (0 of 20) movement cells showed a predictive visual response. The cells with predictive responses never responded to the stimulus when the monkey did not make the saccade that would bring that stimulus into the receptive field, and never discharged in association with that saccade unless it brought a stimulus into the receptive field. The response in the continuous stimulus task was almost always weaker than the visual ON response to a stimulus flashed in the receptive field. Because cells with visual responses but not cells with movement activity alone showed the effect, we conclude that the predictive visual response is a property of the visual processing in the frontal eye field, i.e., a response to the stimulus in the future receptive field. It is not dependent on the actual planning or execution of a saccade to that stimulus. We suggest that the predictive visual mechanism is one in which the brain dynamically calculates the spatial location of objects in terms of desired displacement. This enables the oculomotor system to perform in a spatially accurate manner when there is a dissonance between the retinal location of a target and the saccade necessary to acquire that target. This mechanism does not require an explicit calculation of target position in some supraretinal coordinate system.

Spatial processing in the monkey frontal eye field. II. Memory responses.

Monkeys and humans can easily make accurate saccades to stimuli that appear and disappear before an intervening saccade to a different location. We used the flashed-stimulus task to study the memory processes that enable this behavior, and we found two different kinds of memory responses under these conditions. In the short-term spatial memory response, the monkey fixated, a stimulus appeared for 50 ms outside the neuron's receptive field, and from 200 to 1,000 ms later the monkey made a saccade that brought the receptive field onto the spatial location of the vanished stimulus. Twenty-eight of 48 visuomovement cells and 21/32 visual cells responded significantly under these circumstances even though they did not discharge when the monkey made the same saccade without the stimulus present or when the stimulus appeared and the monkey did not make a saccade that brought its spatial location into the receptive field. Response latencies ranged from 48 ms before the beginning of the saccade (predictive responses) to 272 ms after the beginning of the saccade. After the monkey made a series of 16 saccades that brought a stimulus into the receptive field, 21 neurons demonstrated a longer term, intertrial memory response: they discharged even on trials in which no stimulus appeared at all. This intertrial memory response was usually much weaker than the within-trial memory response, and it often lasted for over 20 trials. We suggest that the frontal eye field maintains a spatially accurate representation of the visual world that is not dependent on constant or continuous visual stimulation, and can last for several minutes.

The rat P450 IID subfamily: complete sequences of four closely linked genes and evidence that gene conversions maintained sequence homogeneity at the heme-binding region of the cytochrome P450 active site.

Four genes in the P450 IID gene subfamily were isolated from Sprague-Dawley rat lambda EMBL 3 and Charon 4A genomic libraries and completely sequenced. Their transcription start sites were determined by primer extension analysis. The four genes designated IID2, IID3, IID4, and IID5 span 4036, 4371, 4678, and 4567 bp, respectively, and are closely linked head to tail on a 60-kb segment of DNA. All IID genes contained nine exons, and interestingly, the IID2, IID3, and IID4 genes possessed an atypical GC5' splice junction in intron 2. All four genes are transcribed, however, IID4 mRNA is produced at a level of less than one-tenth of those of IID2, IID3, and IID5. The exonic regions of these genes displayed from 79 to 84% sequence similarties. Several regions of extremely high nucleotide similarity were found within the introns, exons, and in the flanking regions of the four genes. These localized areas of high nucleotide similarities are the result of former gene conversion events. Of interest was the finding that the most highly similar region of all IID genes that was maintained by gene conversion covers portions of the eighth and ninth exons and the eighth intron. The ninth exon codes for a region of the P450 protein that is well conserved among all P450 gene families and in all species and that is associated with the noncovalently bound heme iron at the enzyme's active site. These data indicate that gene conversions have maintained sequence homogeneity within a critical region of the four P450 IID proteins.

Properties of the recB and recC gene products of Escherichia coli.

The properties of the recB and recC gene products of Escherichia coli were studied using recB and recC gene-inserted plasmids. recB mutants and recC mutants lacked ATP-dependent DNase (recBC enzyme) but showed apparent recovery of enzyme activity on introduction of plasmids carrying the recB and recC gene, respectively. The ATP-dependent DNase was also constructed in vitro by mixing the recB and recC gene products encoded by the plasmids with the corresponding gene. Specific labeling of plasmid-encoded proteins by the maxicell method showed that the recB and recC gene products were 135,000 and 125,000 dalton proteins, respectively. These results suggest that the recB and recC genes are the structural genes of the beta and alpha subunits, respectively, of the recBC enzyme. A gene that encodes a protein of about 100,000 daltons was found to be located between the recB and recC genes. But the product of this gene was shown not to be included in the recBC enzyme.

The rat P450IIE1 gene: complete intron and exon sequence, chromosome mapping, and correlation of developmental expression with specific 5' cytosine demethylation.

The gene coding for the ethanol-inducible, developmentally regulated P450IIE1 was isolated from an lambda EMBL 3 rat genomic library and completely sequenced. The gene spanned 10,373 base pairs and contained nine exons. Upstream and downstream DNA of 1530 and 825 base pairs, respectively, was also sequenced, and the transcription start site was identified by both S1 mapping and primer extension. A typical TATA box was found just upstream of the start site; however, no CCAAT box was apparent. Other repetitive sequences were identified including a partial R.dre.1 sequence upstream of the gene and a long stretch of 160 alternating purines in the second intron. This latter repetitive element is found in many mammalian genes. By use of a panel of mouse-hamster somatic cell hybrids, the P450IIE1 gene was localized to mouse chromosome 7. The hepatic P450IIE1 gene is transcriptionally activated within 1 day after birth and reaches a maximal level of expression at 6 days of age. Using restriction endonuclease sites generated from the gene sequence data and the cytosine methylation-sensitive enzymes HhaI and HpaII, we found that this transcriptional activation during early development is coincident with specific demethylation only at the 5' end of the P450IIE1 gene. Interestingly, other cytosine residues in the middle of the gene became demethylated as rats aged from 1 to 10 weeks, at which time no changes in gene expression occur.