A DNA unwinding factor involved in DNA replication in cell-free extracts of Xenopus eggs.

BACKGROUND: Alteration of chromatin structure is a key step in various aspects of DNA metabolism. DNA unwinding factors such as the high mobility group (HMG) proteins are thought to play a general role in controlling chromatin structure and a specific role in controlling DNA replication. For instance, in the in vitro simian virus 40 replication system, minichromosomes containing HMG-17 replicate more efficiently than those without it, suggesting that HMG-17 enhances the rate of replication of a chromatin template by unfolding the higher-order chromatin structure. At present, however, only limited data suggest an involvement of DNA unwinding factors in DNA replication. RESULTS: We purified from Xenopus eggs a novel heterodimeric factor, termed DNA unwinding factor (DUF), that consists of 87 kDa and 140 kDa polypeptides. DUF unwinds closed-circular duplex DNA in the presence of topoisomerase I, but it does not possess a DNA gyrase activity: it does not introduce negative supercoils into DNA at the expense of ATP hydrolysis. Cloning and sequencing of the cDNAs encoding the two polypeptides revealed that the 87 kDa polypeptide is homologous to a mammalian HMG protein, T160/structure-specific recognition protein. The 140 kDa polypeptide is homologous to yeast Cdc68, a protein that controls the expression of several genes during the G1 phase of the cell cycle by modulating chromatin structure. Immunodepletion of DUF from Xenopus egg extracts drastically reduced the ability of the extract to replicate exogenously added sperm chromatin or plasmid DNA. CONCLUSIONS: We propose that DUF plays a role in DNA replication in Xenopus egg extracts.

DNA synthesis by the isolated nuclear matrix from synchronized plasmodia of Physarum polycephalum.

Nuclear matrices were isolated from plasmodia of a true slime mold, Physarum polycephalum, and the DNA synthetic activity in vitro was examined. These matrices isolated in S-phase catalyzed DNA synthesis requiring Mg2+, deoxyribonucleoside 5'-triphosphates and ATP, without exogenous templates. The activity changed during S-phase with the rate of in vivo DNA replication. Product analysis by gel electrophoresis revealed that the matrices produced Okazaki fragments. These results suggest that DNA synthesis partially reflects in vivo DNA replication. DNA synthesis was sensitive to aphidicolin, heparin and N-ethylmaleimide, indicating involvement of the alpha-like DNA polymerase of Physarum. Exogenous addition of activated DNA stimulated DNA synthesis 4-10-fold and suggested that only some of the existing enzymes are involved in endogenous DNA synthesis. Matrices isolated in G2-phase were also associated with a similar DNA synthetic activity, but they did not produce Okazaki fragments in vitro. It is, therefore, concluded that nuclear matrices are associated with alpha-like DNA polymerase throughout the cell cycle, and that some of the enzymes participate in in vivo DNA replication in S-phase; thus, DNA replication is possibly controlled by this process. The relationship between DNA synthetic activities by the isolated nuclei and matrices was also discussed.

Selective inhibition of DNA polymerase-alpha family with chemically synthesized derivatives of PHYLPA, a unique Physarum lysophosphatidic acid.

PHYLPA, a unique Physarum lysophosphatidic acid (LPA), showed selective inhibition of a family of DNA polymerase alpha, including DNA polymerases alpha, delta and epsilon; but no inhibition of DNA polymerase beta or gamma was observed. To reveal the molecular mechanism of inhibition of DNA polymerases by PHYLPA, four stereoisomers and some other derivatives were synthesized and their effects on DNA polymerases were studied. Among eight derivatives synthesized, PHYLPA-1 (the natural PHYLPA; sodium 1-O-[(9'S,10'R)-9',10'-methanohexadecanoyl]-sn-glycerol 2,3-cyclic phosphate) and PHYLPA-2 (sodium 3-O-[9'S,10'R)-9',10'-methanohexadecanoyl]-sn-glycerol 1,2-cyclic phosphate) were strong and specific inhibitors of a family of DNA polymerase alpha. But their stereoisomers PHYLPA-3 (sodium 1-O-[9'R,10'S)-9',10'-methanohexadecanoyl]-sn-glycerol 2,3-cyclic phosphate) and PHYLPA-4 (sodium 3-O-[9'R,10'S)-9',10'-methanohexadecanoyl-sn-glycerol 1,2 cyclic phosphate) were weak inhibitors, showing the critical importance of stereochemistry of a cyclopropane-containing fatty acid for the inhibitory activity. Some derivatives having no cyclopropane-containing fatty acids--palmitoyl-, oleoyl-, and palmitoleoyl-PHYLPA--showed inhibition to some extent; but 1-palmytoyl and 1-oleoyl lysophosphatidic acid, which has no cyclic phosphate, did not show an apparent inhibitor activity on DNA polymerases. Hence, the extent of the inhibition apparently depends on the stereochemistry of both the fatty acid moiety and the cyclic phosphate.

Differential inhibition of eukaryotic DNA polymerases by halenaquinol sulfate, a p-hydroquinone sulfate obtained from a marine sponge.

Halenaquinol sulfate, a p-hydroquinone sulfate obtained from a marine sponge, inhibited the activity of eukaryotic DNA polymerases in varying degrees; the Ki values for DNA polymerases, alpha, beta, delta and epsilon were 1.3, 80, 17.5 and 2.0 microM, respectively, whereas it was less effective against E. coli DNA polymerase I. The inhibition occurred competitively with each of dATP and dTTP, but non-competitively with dCTP, dGTP and the template DNA. Thus, halenaquinol sulfate is demonstrated to be a potential inhibitor of DNA polymerases alpha and epsilon, and be a useful tool for analyzing the dNTP binding sites of DNA polymerases.

CPP32 activation during dolichyl phosphate-induced apoptosis in U937 leukemia cells.

Treatment of U937 cells with dolichyl phosphate led to an increase in the activity of the ICE family protease CPP32, accompanied with cleavage of pre-CPP32 to generate p17. Peptide inhibitors YVAD-cmk and Z-Asp-CH2-DCB (specific to ICE) and DEVD-CHO (specific to CPP32) blocked the dolichyl phosphate-induced apoptosis. The dolichyl phosphate-induced increase of CPP32 activity was inhibited by adenylate cyclase inhibitors, SQ 22536 and 2',5'-dideoxyadenosine. Dolichyl phosphate caused a transient increase of intracellular cAMP concentration. The results suggest that modulation of cAMP synthesis due to the stimulation of adenylate cyclase by dolichyl phosphate plays a critical role in CPP32 activation and apoptosis.

Increase in DNA polymerase alpha activity associated with DNA synthesis due to FSH or oestrogen in ovaries of immature rats.

DNA polymerase activities and DNA content of ovaries from immature intact rats (4-29 days after birth), hypophysectomized rats and hormone-treated hypophysectomized rats were measured. During normal ovarian growth DNA polymerase alpha activity and DNA content of ovaries increased. The polymerase activity decreased gradually after hypophysectomy without any alteration in the DNA content. Administration of ovine FSH (2 micrograms/day) or oestradiol-17 beta (1 mg/day) to hypophysectomized rats enhanced ovarian DNA content and DNA polymerase alpha activity, whereas DNA polymerase beta activity did not change significantly. These results suggest that DNA polymerase alpha participates in DNA synthesis in these ovaries. The specific activity of DNA polymerase alpha (the activity per microgram DNA) in the ovaries increased between 4 and 14 days after birth, and then remained almost constant; the specific activity declined gradually after hypophysectomy. Administration of FSH or oestradiol-17 beta but not of ovine LH, progesterone or testosterone to hypophysectomized rats restored the specific activity. Mixing experiments with different kinds of ovarian extracts suggested that no activators of DNA polymerase alpha were present in the extracts. These results suggest that FSH or oestrogen causes the induction of DNA polymerase alpha accompanied by DNA synthesis during cell proliferation in ovaries of immature rats.

[Treatment and etiology of spontaneous hemopneumothorax].

The incidence of spontaneous hemopneumothorax is reported to be 1-12% of all cases of spontaneous pneumothorax. We treated 152 cases of spontaneous pneumothorax in the past 8 years and hemopneumothorax occurred in 4 cases which is 2.6% of all cases of spontaneous pneumothorax. All the patients were male and the age ranged from 17 to 30. The total amount of blood loss ranged from 1,200-3,200 mliters and surgical treatment was carried out within 2 days after admission. The bleeding point was visceral pleura of raptured bulla in 2 cases, parietal pleura of the torn adhesion in 1 case, and both visceral and parietal pleura in 1 case. Postoperative course was satisfactory and discharged within 2 weeks after admission in all cases. The authors concluded that early thoracotomy is recommended for spontaneous hemopneumothorax.

[A case of paraesophageal bronchogenic cyst with esophageal communication].

A case of mediastinal bronchogenic cyst communicating with the esophagus was reported. Previously, only 2 cases have been reported in the available literature. A 34-year-old man was admitted with a cystic mass communicating with the esophagus which was demonstrated on a barium study. Operation was performed with a suspect of esophageal diverticulum or congenital cyst with esophageal communication. At right thoracotomy, a mass measuring 6.0 x 5.0 cm with a well-defined patent communication to the esophagus was resected. It was a monolocular cyst containing a small amount of viscous mucus. Histologically, the cyst lined by a ciliated columnar epithelium, and it was diagnosed as a bronchogenic cyst because of the presence of the mucous glands, smooth muscle tissue and cartilage. This is the first case report of mediastinal bronchogenic cyst with esophageal communication appeared in the Japanese literature.

Infected Charcot spine.

STUDY DESIGN: Case report of an infected Charcot spine following spinal cord injury. OBJECTIVE: To describe this very rare pathological condition and the results of surgical treatment. SETTING: A department of orthopaedic surgery in Japan. METHODS: A 44-year-old man presented with a destructive lesion in the lumbo-sacral spine and a fistula in his back. Anterior bone graft, percutaneous external spinal fixation, and suction/irrigation of the wound were performed. After 4 months, posterior spinal instrumentation surgery was carried out. RESULTS: Primary closure of the fistula and complete bone fusion was achieved after the operation. CONCLUSION: Infection of a Charcot spine, although a rare clinical entity, should be considered as a diagnostic possibility in the spinal cord-injured patients. External spinal fixation is a useful method for the unstable spinal lesion with infection.

[Intradiscal injection of hypertonic saline, phenol-glycerin and osmic acid for the treatment of lumbar disc herniation: an experimental study].

UNLABELLED: The present study was designed to investigate the possible clinical application of hypertonic saline (HS), phenol in glycerin (PHG) and osmic acid (OSA) for intradiscal therapy. MATERIALS & METHODS: HS in several concentrations, 10% PHG and 4% OSA were separately injected into the lumbar intervertebral discs of 60 Japanese white rabbits. Additionally, these substances were placed directly on the dura of the spinal cord of 48 guinea pigs. The animals were sacrificed periodically and were submitted to histological examination using light microscopy. RESULTS: HS caused localized necrosis of the nucleus pulposus cells in a concentration-related fashion. Some discs decreased their height. With time, all the discs generally regained their normal histology. Following administration of 10% PHG, the area of necrosis of the nucleus pulposus cells was more extensive than that by HS, but the regenerative or reparative reaction was not so brisk. Examination of the discs treated with 4% OSA demonstrated severe changes in the nucleus pulposus and the inner annulus fibrosus with resultant disc-space narrowing. The reparative tissue seen after injection of OSA was fibrocartilage in nature. No histological change was seen in the surrounding tissue including the neural tissue following administration of any of the substances. DISCUSSION: Chymopapain is the substance most frequently used for clinical chemonucleolysis. The major clinical complication with chymopapain has been anaphylaxis. The present substances have been used in other clinical applications without reports of anaphylaxis. In this report, HS was shown to hold the potential for reducing intradiscal pressure without induction of scar tissue or significant loss of disc function. PHG and OSA caused considerable but circumscribed histological damage to the disc tissue, but had no such effect on the neural tissues. These data suggested that HS, PHG and OSA may have clinical applications as agents in intradiscal therapy.

Evidence of the existence of a high molecular weight form of DNA polymerase alpha in sea urchin eggs.

DNA polymerase alpha combined with the endoplasmic reticulum (ER) was isolated from unfertilized sea urchin eggs. NaCl treatment of this fraction released DNA polymerase alpha from the ER. The molecular size (the S value) of the ER-free DNA polymerase alpha changed with the concentration of NaCl used; being 23 S, 11-15 S and 6-8 S in the presence of 0.05-0.12 M, 0.12-0.24 M and more than 0.24 M NaCl. DNA polymerase alpha activity decreased concomitantly with the reduction in molecular size. The 6-8 S form of DNA polymerase alpha did not aggregate by itself nor with other cellular components nonspecifically, when the 23 S form was present. These results are evidence of the presence of 6-8 S DNA polymerase alpha as a high molecular weight form (23 S-form) in sea urchin eggs.

DNA synthesis in vitro with an endoplasmic-reticulum-DNA-polymerase complex from unfertilized sea urchin eggs.

An endoplasmic-reticulum-DNA-polymerase complex was prepared from unfertilized sea urchin eggs and its DNA-synthesizing activity was examined using single-stranded DNA of bacteriophage fd as a template. The complex catalyzed the ribonucleotide-dependent DNA synthesis which required dNTPs, NTPs, Mg2+ and single-stranded DNA. The DNA synthesis was sensitive to aphidicolin and N-ethylmaleimide but was resistant to 2',3'-dideoxyribosylthymine 5'-triphosphate (ddTTP) and alpha-amanitin, suggesting the involvement of DNA polymerase alpha. In parallel with the DNA synthesis, a small amount of RNA was synthesized in the presence of 100 micrograms/ml alpha-amanitin. The Km value of ribonucleotides for the RNA synthesis coincided with that for the DNA synthesis, suggesting a correlation between the DNA and RNA syntheses. Labelling of the products with [gamma-32P]ATP followed by DNA digestion with pancreatic DNase I revealed the attachment of an oligoribonucleotide (7-11 bases in length) at the 5' ends of the DNA products. These observations suggest that in DNA synthesis, primer RNA synthesis occurs first, followed by DNA chain elongation. During 1-90-min incubation, the amount of the DNA synthesized increased but the length was not significantly increased. Over 80% of the number of synthesized DNA molecules comprised a single population of short DNA fragments (60-200 bases, on average 120 bases in length) and the number of fragments increased, depending on the incubation time. However, DNA fragments of various sizes (about 100-6000 bases) were synthesized with DNA polymerase alpha solubilized from the endoplasmic-reticulum-DNA-polymerase complex. All this evidence suggests that in vitro, the complex preferentially synthesizes a particular size of short DNA fragments. The significance of the fragments is discussed.

Subcellular localization of DNA polymerase gamma and changes in its activity in sea urchin embryos.

1. Subcellular localization and changes in the activity of DNA polymerase gamma were examined in sea urchin eggs and embryos. 2. The enzyme was shown to be localized predominantly in mitochondria by differential and isopycnic centrifugation. 3. During embryogenesis, the enzyme activity per embryo remained constant until blastula stage, and thereafter increased. 4. Similarly mitochondrial DNA per embryo increased, indicating that mitochondrial DNA replication starts during embryogenesis. 5. The gamma-activity per mitochondrial DNA remained constant during embryogenesis. 6. These results suggest that mitochondria contain a constant amount of replicative enzyme (DNA polymerase gamma) regardless of mitochondrial DNA replication, which differs from the case of nuclear DNA replication.

The possible involvement of replication-related proteins with a DEAD-box-like motif in cell-free DNA replication of Xenopus eggs.

Two types of antibodies were prepared: one directed against an oligopeptide specific to P1 protein, a mammalian homologue of yeast MCM3, and the other against an oligopeptide with a DEAD box motif, which is a highly conserved sequence in the P1 protein family. Immunoprecipitation of the eluate from anti-P1 family IgG-bound beads, which had been incubated in Xenopus egg extracts, with anti-P1 IgG-bound beads revealed that three proteins were coprecipitated. Two proteins remained in the supernatant after the immunoprecipitation of the eluate from anti-P1 family IgG-bound beads with anti-P1 IgG-bound beads. The immunodepleted extracts with anti-P1 family IgG-bound beads showed much lower DNA replication activity than did mock-treated extracts. Recovery of replication was achieved by supplementing the depleted extracts with both the eluate from anti-P1 IgG-bound beads and the supernatant obtained after the immunoprecipitation of the eluate with anti-P1 IgG-bound beads but not by supplementing the extracts with only the proteins eluted from anti-P1 IgG-bound beads. These findings suggest that some proteins containing a DEAD-box-like motif as well as mammalian homologues of yeast MCM2, MCM3 and CDC46 play an important role in cell-free DNA replication of Xenopus eggs.

Follicle-stimulating hormone and estrogen elevate deoxyribonucleic acid alpha-nucleotidyltransferase activity in relationship to deoxyribonucleic acid synthesis in immature rat ovaries.

The deoxyribonucleic acid polymerase activities and deoxyribonucleic acid contents of ovaries were measured to clarify the relationship between hormone-stimulated cell proliferation and the enzymes in ovaries of immature intact rats (4 to 29 days after birth) and hypophysectomized rats. The specific activity of deoxyribonucleic acid alpha-nucleotidyltransferase (the activity per microgram of deoxyribonucleic acid of the ovaries) drastically increased with an increase in the deoxyribonucleic acid content of the ovaries from 4 to 14 days after birth and then remained constant or slightly decreased after the increase, while the activity decreased gradually after hypophysectomy with no increase in the DNA content. Ovine follicle-stimulating hormone or estradiol-17 beta enhanced the deoxyribonucleic acid synthesis and alpha-nucleotidyltransferase activity in the ovaries of hypophysectomized rats, while deoxyribonucleic acid beta-nucleotidyltransferase activity showed no significant change. Ovine luteinizing hormone, progesterone, and testosterone caused no significant increases. These results suggest that follicle-stimulating hormone or estrogen causes the induction of deoxyribonucleic acid alpha-nucleotidyltransferase accompanied by deoxyribonucleic acid synthesis in cell proliferation in immature rat ovaries.

Localization of DNA polymerase alpha on the nuclear membrane in sea urchin embryos.

Subnuclear localization of DNA polymerase alpha was studied in sea urchin embryos. Blastula nuclei treated with EDTA and potassium phosphate released subnuclear components bearing most of the nuclear DNA polymerase alpha. These components were suggested to be a part of nuclear membrane based on their buoyant densities (1.177 and 1.136 g/cm3) in isopyknic centrifugation and the nuclear pore-like structure. Contamination with DNA and endoplasmic reticulum membrane to the subnuclear components was shown to be negligible. These results suggested that DNA polymerase alpha associates with nuclear membrane of sea urchin embryos. Nuclear membrane deprived of DNA polymerase alpha was able to associate with nuclear DNA polymerase alpha from blastulae and the cytoplasmic enzyme of unfertilized eggs efficiently, but not with the cytoplasmic enzyme of gastrulae. This result suggests that the nuclear membrane is originates from the endoplasmic reticulum with which DNA polymerase alpha associates in unfertilized eggs.

Analysis of replicating DNA molecules from embryos of the sea urchin, Hemicentrotus pulcherrimus, by electron microscopy.

DNA was extracted from embryos of the sea urchin, Hemicentrotus pulcherrimus, at the S phase and examined by electron microscopy. We detected replication microbubbles with a mean size of 404 bases, in addition to replication macrobubbles of more than 1.0 kilobase (kb) in length. Seventy-five percent of the center-to-center distances of the microbubbles were 0.6-1.8 kb with a mean of 1.2 kb. Forty-five percent of the microbubbles were arranged as clusters of four or five microbubbles. These results suggest that at least 34% of the initiation sites for DNA replication are present on a DNA molecule in clusters in which the sites are arranged at 1.2-kb intervals.