The archaeal DNA primase: biochemical characterization of the p41-p46 complex from Pyrococcus furiosus.

We characterized the primase complex of the hyperthermophilic archaeon, Pyrococcus furiosus. The two proteins, Pfup41 and Pfup46, have similar sequences to the p48 and p58 subunits, respectively, of the eukaryotic DNA polymerase alpha-primase complex. Unlike previously reported primases, the Pfup41 preferentially utilizes deoxyribonucleotides for its de novo synthesis, and moreover, it synthesizes up to several kilobases in length in a template-dependent manner (Bocquier, A., Liu, L., Cann, I., Komori, K., Kohda, D., and Ishino, Y. (2001) Curr. Biol. 11, 452-456). The p41-p46 complex showed higher DNA binding activity than the catalytic p41 subunit alone. In addition, the amount of DNA synthesized by the p41-p46 complex was much more abundant and shorter in length than that by Pfup41 alone. The activity for RNA primer synthesis, which was not detected with Pfup41, was observed from the reaction using the p41-p46 complex in vitro. The in vitro replication of M13 single-stranded DNA by the P. furiosus proteins was stimulated by ATP. Observation of the labeled primers by using [gamma-(32)P]ATP in the substrates suggests ATP as the preferable initiating nucleotide for the p41-p46 complex. These results show that the primer synthesis activity of Pfup41 is regulated by Pfup46, and the p41-p46 complex may function as the primase in the DNA replication machinery of P. furiosus, in a similar fashion to the eukaryotic polymerase alpha-primase complex.

Atomic structure of the clamp loader small subunit from Pyrococcus furiosus.

In eukaryotic DNA replication, replication factor-C (RFC) acts as the clamp loader, which correctly installs the sliding clamp onto DNA strands at replication forks. The eukaryotic RFC is a complex consisting of one large and four small subunits. We have determined the crystal structure of the clamp loader small subunit (RFCS) from Pyrococcus furiosus. The six subunits, of which four bind ADP in their canonical nucleotide binding clefts, assemble into a dimer of semicircular trimers. The crescent-like architecture of each subunit formed by the three domains resembles that of the delta' subunit of the E. coli clamp loader. The trimeric architecture of archaeal RFCS, with its mobile N-terminal domains, involves intersubunit interactions that may be conserved in eukaryotic functional complexes.

Functional interactions of an archaeal sliding clamp with mammalian clamp loader and DNA polymerase delta.

BACKGROUND: By the total genome sequencing of several archaeal organisms, it has been confirmed that many archaeal proteins related to genetic information systems, including DNA replication, transcription and translation, have similar sequences to those of eukaryotes. In eukaryotic DNA replication, proliferating cell nuclear antigen (PCNA) works in clamping DNA polymerases on the DNA template and accomplishes a processive DNA synthesis. Archaea encode PCNA homologues in their genomes and Pyrococcus furiosus PCNA (PfuPCNA) stimulates the DNA synthesizing activities of the DNA polymerases, Pol I and Pol II, in this organism. RESULTS: We have demonstrated that PfuPCNA interacts functionally with calf thymus DNA polymerase delta (Pol delta) and stimulates its activity. Moreover, human replication factor C (RFC) enhances the PfuPCNA-dependent DNA synthesis activity of Pol delta, indicating that human RFC works as the clamp loader for PfuPCNA. These results showed that the three-dimensional structures of archaral PCNA and RFC are actually similar enough to their eukaryotic counterparts to allow a molecular substitution between the two biological domains, albeit at a lower efficiency. CONCLUSIONS: We found that the archaeal molecule interacts functionally with the eukaryotic members in the DNA replication process. This finding supports the idea that studies on the DNA replication mechanism of archaeal organisms will provide many important clues for understanding of the intricate molecular recognition that is inherent to the DNA replication machinery in Eukarya.

[Hemodynamic effects of STA-MCA anastomosis on patients with occlusion of the main cerebral artery].

PURPOSE: We studied cerebral circulation in patients with occlusion of the main cerebral artery and investigated the efficacy of STA-MCA anastomosis. PATIENTS AND METHODS: Thirty-six patients with occlusion of the main cerebral artery were studied. Twenty-three patients had occlusion of the internal carotid artery and 13 had occlusion of the middle cerebral artery. The mean age was 62 years. Cerebral blood flow (CBF) was measured in all patients and cerebrovascular reactivity (CVR) was examined in 11 patients by xenon enhanced CT. Intraoperatively, cortical arterial pressure and anastomotic flow were measured. RESULTS: There was no perioperative mortality or morbidity. There was no ipsilateral stroke recurrence during the follow-up period averaging 35.1 months. Patency of the anastomosis was verified in 91% of the patients by magnetic resonance angiography. Twenty-three (64%) patients showed decreased CBF before the operation and 57% of these patients showed improvement to the normal range after STA-MCA anastomosis. All of the eight patients with decreased CVR showed improvement after the operation. Anastomotic flow correlated significantly with the cortical arterial pressure. CONCLUSION: STA-MCA anastomosis could improve cerebral circulation of patients with low CBF or low CVR due to occlusion of the main cerebral arterial. It was concluded that STA-MCA anastomosis may contribute to the reduction of stroke recurrence, if perioperative complications are reduced.

Biochemical analysis of replication factor C from the hyperthermophilic archaeon Pyrococcus furiosus.

Replication factor C (RFC) and proliferating cell nuclear antigen (PCNA) are accessory proteins essential for processive DNA synthesis in the domain Eucarya. The function of RFC is to load PCNA, a processivity factor of eukaryotic DNA polymerases delta and epsilon, onto primed DNA templates. RFC-like genes, arranged in tandem in the Pyrococcus furiosus genome, were cloned and expressed individually in Escherichia coli cells to determine their roles in DNA synthesis. The P. furiosus RFC (PfuRFC) consists of a small subunit (RFCS) and a large subunit (RFCL). Highly purified RFCS possesses an ATPase activity, which was stimulated up to twofold in the presence of both single-stranded DNA (ssDNA) and P. furiosus PCNA (PfuPCNA). The ATPase activity of PfuRFC itself was as strong as that of RFCS. However, in the presence of PfuPCNA and ssDNA, PfuRFC exhibited a 10-fold increase in ATPase activity under the same conditions. RFCL formed very large complexes by itself and had an extremely weak ATPase activity, which was not stimulated by PfuPCNA and DNA. The PfuRFC stimulated PfuPCNA-dependent DNA synthesis by both polymerase I and polymerase II from P. furiosus. We propose that PfuRFC is required for efficient loading of PfuPCNA and that the role of RFC in processive DNA synthesis is conserved in Archaea and Eucarya.

Scanning electron microscopy description of a new species of Demodex canis spp.

Between 1997 and 1999, the prevalence of Demodex canis mites was determined in 150 dogs. In two dogs, we found two different species of mites; Demodex canis and another, unidentified, Demodex mite. The unidentified Demodex mite species had several different morphological features. First, it had a short opisthosoma and an obtuse end. In addition, the fourth coxisternal plate was rectangular and there was a band-like segmental plate between the fourth coxisternal plate and opisthosoma. Although all of the morphology and the development of male mites could not be investigated in this study, the location of the opisthosoma and the genital pore clearly differed from Demodex canis, suggesting that this unidentified mite is a new species.

Effects of acupuncture on peripheral T lymphocyte subpopulation and amounts of cerebral catecholamines in mice.

The aim of this study was to investigate the effects of acupuncture on peripheral lymphocyte subpopulations and cerebral catecholamines. In order to examine the effects of acupuncture, two experiments were performed. Experiment 1: Eighteen female mice (strain; C57BL/6) at the age of 7 weeks were divided three groups, (a) sham operated (control; n=6), (b) ovariectomized (OVX; n=6), and (c) ovariectomized and stimulated by subcutaneous needles on acupuncture point, Shenshu (BL23) at the both sides of the back for 20 days (OVX+Acu; n=6). These animals were sacrificed at 20 days after needle insertion, and the splenic lymphoid cells were examined by two-color flow cytometry, using monoclonal antibodies (mAb) to the cell surface antigens, CD3, CD4, CD8a and NK1.1 (CD56). In the ovariectomized (OVX) group, the peripheral CD4/CD8 ratio was significantly increased and the ratio of natural killer (NK) cells (CD3-NK1.1+; CD3 negative, NK1.1 positive) to T lymphocytes was decreased compared to the sham control group. In the ovariectomized with needle insertion (OVX+Acu) group, the CD4/CD8 ratio was reduced, but the NK cells ratio was not changed compared to the OVX group. Experiment 2: To investigate the acute effects of subcutaneous needle insertion, male C57BL/6 mice (7 weeks old) were used (n=6, each group). The acupuncture points Shen-shu (BL23) on the backs of the male mice were also stimulated by subcutaneous needles for 3 and 7 days. As a result, the CD4/CD8 ratio was significantly decreased at day 3 and day 7, compared to the control group. On the other hand the NK cells ratio and activated T-cells were increased at day 7. The mitogenic activities in the splenic lymphocytes were also increased by acupuncture stimulation at day 3. Catecholamine contents in the hippocampus were measured by high performance liquid chromatography with the electro-chemical detector (ECD-HPLC) method. No significant change was observed in either dopamine contents or norepinephrine; however, dopamine metabolite, homovanillic acid (HVA) and DOPAC (3,4-dihydroxyphenylacetic acid) were increased at day 3. The study suggests that acupuncture has effects on peripheral lymphocyte subpopulations and may modulate mitogenic activity. In addition, acupuncture may stimulate dopamine turnover.

Functional interactions of a homolog of proliferating cell nuclear antigen with DNA polymerases in Archaea.

Proliferating cell nuclear antigen (PCNA) is an essential component of the DNA replication and repair machinery in the domain Eucarya. We cloned the gene encoding a PCNA homolog (PfuPCNA) from an euryarchaeote, Pyrococcus furiosus, expressed it in Escherichia coli, and characterized the biochemical properties of the gene product. The protein PfuPCNA stimulated the in vitro primer extension abilities of polymerase (Pol) I and Pol II, which are the two DNA polymerases identified in this organism to date. An immunological experiment showed that PfuPCNA interacts with both Pol I and Pol II. Pol I is a single polypeptide with a sequence similar to that of family B (alpha-like) DNA polymerases, while Pol II is a heterodimer. PfuPCNA interacted with DP2, the catalytic subunit of the heterodimeric complex. These results strongly support the idea that the PCNA homolog works as a sliding clamp of DNA polymerases in P. furiosus, and the basic mechanism for the processive DNA synthesis is conserved in the domains Bacteria, Eucarya, and Archaea. The stimulatory effect of PfuPCNA on the DNA synthesis was observed by using a circular DNA template without the clamp loader (replication factor C [RFC]) in both Pol I and Pol II reactions in contrast to the case of eukaryotic organisms, which are known to require the RFC to open the ring structure of PCNA prior to loading onto a circular DNA. Because RFC homologs have been found in the archaeal genomes, they may permit more efficient stimulation of DNA synthesis by archaeal DNA polymerases in the presence of PCNA. This is the first stage in elucidating the archaeal DNA replication mechanism.

Two family B DNA polymerases from Aeropyrum pernix, an aerobic hyperthermophilic crenarchaeote.

DNA polymerase activities in fractionated cell extract of Aeropyrum pernix, a hyperthermophilic crenarchaeote, were investigated. Aphidicolin-sensitive (fraction I) and aphidicolin-resistant (fraction II) activities were detected. The activity in fraction I was more heat stable than that in fraction II. Two different genes (polA and polB) encoding family B DNA polymerases were cloned from the organism by PCR using degenerated primers based on the two conserved motifs (motif A and B). The deduced amino acid sequences from their entire coding regions contained all of the motifs identified in family B DNA polymerases for 3'-->5' exonuclease and polymerase activities. The product of polA gene (Pol I) was aphidicolin resistant and heat stable up to 80 degrees C. In contrast, the product of polB gene (Pol II) was aphidicolin sensitive and stable at 95 degrees C. These properties of Pol I and Pol II are similar to those of fractions II and I, respectively, and moreover, those of Pol I and Pol II of Pyrodictium occultum. The deduced amino acid sequence of A. pernix Pol I exhibited the highest identities to archaeal family B DNA polymerase homologs found only in the crenarchaeotes (group I), while Pol II exhibited identities to homologs found in both euryarchaeotes and crenarchaeotes (group II). These results provide further evidence that the subdomain Crenarchaeota has two family B DNA polymerases. Furthermore, at least two DNA polymerases work in the crenarchaeal cells, as found in euryarchaeotes, which contain one family B DNA polymerase and one heterodimeric DNA polymerase of a novel family.

Induction of apoptosis with fusarenon-X in mouse thymocytes.

The effects of fusarenon-X (12,13-epoxytrichothecene; FX) on mouse thymus and T-cell subpopulations were studied. In mice that received three intraperitoneal injections of FX, the thymus showed severe atrophy, the thymic cortex almost completely disappeared, and the total number of thymocytes decreased to 2.2% of that of normal mice. CD4+ CD8+ thymocytes were almost completely depleted by this treatment while CD4+ CD8-, CD4- CD8+ and CD4- CD8- thymocytes were not reduced to such an extent, suggesting that selective damage in CD4+ CD8+ thymocytes was induced by FX. In spleen, CD4+ or CD8+ lymphocytes and CD4- CD8- non-T cells remained unchanged. Next, the mode of damage in thymocytes was investigated by a single injection with FX. The lymphocyte nuclei were fragmented and positive for TUNEL (TdT-mediated dUTP nick-end labeling) staining in the thymic cortex 20 h after FX injection. By electron microscopy, apoptotic lymphocytes with condensed nuclei and stroma cells ingesting many nuclear fragments were frequently observed in the thymic cortex. Internucleosomal DNA fragmentation was apparent in the thymocytes treated with FX both in vivo and in vitro. Thus, we demonstrated that the trichothecene mycotoxin FX is a new cause of apoptosis in CD4+ CD8+ thymocytes of mice besides the other factors that cause similar effects.

Loss of material from chromosome arm 1p during malignant progression of meningioma revealed by fluorescent in situ hybridization.

BACKGROUND: Atypical and anaplastic meningiomas tend to recur and to invade adjacent brain, bone, and skin. They also can metastasize to extracranial organs such as the lung, liver, or bone, causing death. Recent reports have indicated that allelic deletion of chromosome 1p is associated with malignant progression of meningiomas. METHODS: Cytogenetic analysis of 37 meningiomas was performed using double-target fluorescent in situ hybridization (FISH) and focusing on chromosome arm 1p. The meningioma series included 17 benign meningiomas, 11 atypical meningiomas, and 9 anaplastic meningiomas. FISH was performed with pericentromeric (1q12) and subtelomeric (1p36) DNA probes to cell nuclei prepared from surgically extirpated tumor samples. RESULTS: A high incidence of deletion of at least part of 1p was observed in 60.0% of atypical and 85.7% of anaplastic meningiomas. Furthermore, statistically significant differences were found with respect to these data between benign versus atypical/anaplastic meningiomas. In four cases both primary and recurrent tumors from the same patient also were investigated for allelic status. CONCLUSIONS: The results of the current study support the existence of tumor suppressor gene(s) on 1p associated with malignant progression of meningioma, and suggest that detection of the allelic status of chromosome 1p by FISH may assist physicians in predicting the clinical prognosis of patients affected by this type of brain tumor.

Rapid induction of lymphoid leukosis and ascites by avian leukosis virus from a lymphoid leukosis cell line.

To examine whether a lymphoid leukosis (LL) cell line releases an LL-specific avian leukosis virus (ALV) or not, two viral materials, culture fluid and a concentrated viral material from an LL-cell line, were inoculated into a total of 74 day-old chicks of line 15I in 5 experiments. Spectrum of diseases induced, their incidence and incubation periods to onset were examined. Fifteen chicks were inoculated with the culture fluid and 9 (60%) developed ascites [59-119 days post inoculation (dpi); geometric mean (GM) of dpi, GM: 89.6)], but LL was not induced in any chicks inoculated. Fifty-nine chicks were inoculated with the concentrated viral material and LL was recognized in 13 (22.0%) (27-74 dpi; GM: 48.4), ascites with LL in 11 (18.6%) (34-75 dpi; GM: 41.3), ascites alone in 21 (35.6%) (32-83 dpi; GM: 48.2), erythroblastosis in 2 (3.4%) (70-102 dpi; GM: 84.5), and other diseases in 12 (20.3%) (43-102 dpi; GM: 61.8). LL lesions were frequently observed in the liver, spleen, kidneys, bursa of Fabricius (bursa), bone marrow and gonads. Mild lymphocytic foci in some visceral organs and perivascular cuffing in the central nervous system were observed mainly in several chicks diagnosed as having complication of ascites with LL or other diseases. In addition to these lesions, atrophy of bursa and thymuses was recognized in them. No antibodies against Marek's disease virus (MDV) and reticuloendotheliosis virus were detected in 36 sera taken from the chicks inoculated with the concentrated viral material. Serotype 2 MDV was isolated from the buffy coat of some inoculated chicks. These results suggest that the properties of ALV inoculated and immunosuppression caused by inoculation with high doses of ALV are involved in rapid induction of LL and expression of pathogenicity of serotype 2 MDV released from the LL cell line and included in the viral inoculum. This is the first report describing the rapid induction of LL and ascites in chicks.

Construction of catalase deficient Escherichia coli strains for the production of uricase.

To produce catalase-free uricase preparations, we constructed catalase-deficient strains from Escherichai coli MC1000 and MM294 and used them as recombinant host strains. The parent strains and catalase-deficient strains showed no differences in the growth characteristics by shaking culture in Erlenmeyer flasks. The catalase deficient strain derived from MC1000 transformed with the uricase expression plasmid pUT118 (strain SN0037) had growth characteristics and the uricase productivity comparable to those of the parent host strain MC1000 in fed-batch culture in a jar fermentor and no catalase activity was detected in cell-free extracts. However, the katG disrupted strains from MM294 carrying pUT118 had poor growth and their uricase productivities were low compared to those of the parent strain MM294. Using the strain SN0037, a catalase-free uricase preparation was obtained with fewer purification procedures and the final recovery of uricase activity was improved. The catalase-deficient E. coli host strain will be a suitable host for the production of the uricase, free of catalase activity, in high yield.

Cloning, purification, and properties of a cofactor-independent glutamate racemase from Lactobacillus brevis ATCC 8287.

A glutamate racemase gene of Lactobacillus brevis ATCC 8287 was cloned into Escherichia coli TM93 by the phenotypic complementation of a phosphoenolpyruvate carboxylase deficiency on minimum agar medium containing D-glutamate. The gene was localized to a 1.4-kb HindIII-EcoRI DNA fragment and the total nucleotide sequence of the fragment was analyzed. The gene has typical promoter and SD sequences which appeared to function in E. coli. The deduced amino acid sequence of the enzyme had 276 amino acids and the molecular weight was calculated as 29,426. Two cysteine residues and their surrounding regions of the enzyme are homologous to those of other cofactor-independent racemases. The glutamate racemase was purified from recombinant E. coli to homogeneity and characterized. The enzyme required no cofactors for the activity, and retained its activity even in 2 M (300 g/l) L-glutamate.

[Alexia-agraphia of kanji (Japanese morphogram) after left posterior-inferior temporal lesion]. / Alexie-agraphie pour l'écriture kanji après lésion temporale postéro-inférieure gauche.

Several cases of selective alexia with agraphia of kanji have been reported in Japan in this decade. It is well known that the lesion in the posterior inferior temporal lobe of the dominant hemisphere is responsible for this cognitive syndrome. Neuropsychological data in our patient suggest that the postero-inferior region of the temporal lobe of the dominant hemisphere may be the visuo-verbal association area for the analysis of the complex visuo-verbal information. The symptoms caused by the same lesion in western patients might be subangular alexia (alexia without agraphia). Alexia with agraphia of kanji and subangular alexia would appear to be distinct entities, but a dual processing hypothesis of visuo-verbal information and the concept of the visuo-verbal association area might well explain both syndromes.

Effects of the amplification of the genes coding for the L-threonine biosynthetic enzymes on the L-threonine production from methanol by a gram-negative obligate methylotroph, Methylobacillus glycogenes.

We constructed recombinant plasmids carrying the genes coding for the L-threonine biosynthetic enzymes, the hom gene, the hom-thrC genes, and the thrB genes, of a gram-negative obligate methylotroph, Methylobacillus glycogenes, and examined the effects of them on the production of L-threonine from methanol. The hom gene, which encodes the homoserine dehydrogenase, and the hom-thrC genes, containing the gene coding for threonine synthase together with the hom gene, were cloned from a wild-type strain, and the thrB gene encoding the desensitized homoserine kinase was cloned from an L-threonine-producing mutant, ATR80. The recombinant plasmids were transferred into ATR80 and its L-isoleucine auxotroph, A513, by conjugation. Amplification of the genes coding for the L-threonine biosynthetic enzymes elevated the activities of the L-threonine biosynthetic enzymes of the transconjugants 10- to 30-fold over those of the strains containing only vectors. The L-threonine production from methanol in test-tube cultivation was increased about 30% and 40% by the amplification of the hom gene and the hom-thrC gene respectively, and it was slightly increased by that of the thrB gene. The effects of gene amplification were confirmed by the cultivation in 5-1 jar fermentors. The best producer, an A513 transconjugant containing the plasmid carrying the hom-thrC genes, produced 16.3 g/l L-threonine for 72 h.

Cloning and nucleotide sequences of the homoserine dehydrogenase genes (hom) and the threonine synthase genes (thrC) of the gram-negative obligate methylotroph Methylobacillus glycogenes.

We have cloned the homoserine dehydrogenase genes (hom) from the gram-negative obligate methylotrophs Methylobacillus glycogenes ATCC 21276 and ATCC 21371 by complementation of an Escherichia coli homoserine dehydrogenase-deficient mutant. The 4.15-kb DNA fragment cloned from M. glycogenes ATCC 21371 also complemented an E. coli threonine synthase-deficient mutant, suggesting the DNA fragment contained the thrC gene in addition to the hom gene. The homoserine dehydrogenases expressed in the E. coli recombinants were hardly inhibited by L-threonine, L-phenylalanine, or L-methionine. However, they became sensitive to the amino acids after storage at 4 degrees C for 4 days as in M. glycogenes. The structures of the homoserine dehydrogenases overexpressed in E. coli were thought to be different from those in M. glycogenes, probably in subunit numbers of the enzyme, and were thought to have converted to the correct structures during the storage. The nucleotide sequences of the hom and thrC genes were determined. The hom genes of M. glycogenes ATCC 21276 and ATCC 21371 encode peptides with M(r)s of 48,225 and 44,815, respectively. The thrC genes were located 50 bp downstream of the hom genes. The thrC gene of ATCC 21371 encodes a peptide with an M(r) of 52,111, and the gene product of ATCC 21276 was truncated. Northern (RNA) blot analysis suggests that the hom and thrC genes are organized in an operon. Significant homology between the predicted amino acid sequences of the hom and thrC genes and those from other microorganisms was found.

Lymphoplasmacytic lymphoma in a stallion.

Lymphoplasmacytic lymphoma found in a 6-year-old Anglo-Arabian stallion was investigated histologically, immunohistochemically and ultrastructurally. The animal showed a large mediastinal mass and generalized lymph node involvement. The neoplastic cells were in various differentiation stages of small lymphocyte, centrocyte, centroblast, immunoblast and plasma cell. Some neoplastic cells showed positive cytoplasmic reactivity for mu and lambda chains. There were well developed rough endoplasmic reticulum (RER) and Golgi complexes in plasmacytoid cells, and slightly developed RER or a few long strands of RER in medium-sized to large lymphoid cells. These findings suggest that this neoplasm is of B-cell origin.