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Human origin recognition complex (hORC) binds to the DNA replication origin and then initiates DNA replication. However, hORC does not exhibit DNA sequence-specificity and how hORC recognizes the replication origin on genomic DNA remains elusive. Previously, we found that hORC recognizes G-quadruplex structures potentially formed near the replication origin. Then, we showed that hORC subunit 1 (hORC1) preferentially binds to G-quadruplex DNAs using a hORC1 construct comprising residues 413 to 511 (hORC1 413-511 ). Here, we investigate the structural characteristics of hORC1 413-511 in its free and complex forms with G-quadruplex DNAs. Circular dichroism and nuclear magnetic resonance (NMR) spectroscopic studies indicated that hORC1 413-511 is disordered except for a short α-helical region in both the free and complex forms. NMR chemical shift perturbation (CSP) analysis suggested that basic residues, arginines and lysines, and polar residues, serines and threonines, are involved in the G-quadruplex DNA binding. Then, this was confirmed by mutation analysis. Interestingly, CSP analysis indicated that hORC1 413-511 binds to both parallel- and (3 + 1)-type G-quadruplex DNAs using the same residues, and thereby in the same manner. Our study suggests that hORC1 uses its intrinsically disordered G-quadruplex binding region to recognize parallel-type and (3 + 1)-type G-quadruplex structures at replication origin., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Cells must coordinate the activation of thousands of replication origins dispersed throughout their genome. Active transcription is known to favor the formation of mammalian origins, although the role that RNA plays in this process remains unclear. We show that the ORC1 subunit of the human Origin Recognition Complex interacts with RNAs transcribed from genes with origins in their transcription start sites (TSSs), displaying a positive correlation between RNA binding and origin activity. RNA depletion, or the use of ORC1 RNA-binding mutant, result in inefficient activation of proximal origins, linked to impaired ORC1 chromatin release. ORC1 RNA binding activity resides in its intrinsically disordered region, involved in intra- and inter-molecular interactions, regulation by phosphorylation, and phase-separation. We show that RNA binding favors ORC1 chromatin release, by regulating its phosphorylation and subsequent degradation. Our results unveil a non-coding function of RNA as a dynamic component of the chromatin, orchestrating the activation of replication origins., (© 2023. The Author(s).)

Origin recognition complex (ORC) binds to replication origins in eukaryotic DNAs and plays an important role in replication. Although yeast ORC is known to sequence-specifically bind to a replication origin, how human ORC recognizes a replication origin remains unknown. Previous genome-wide studies revealed that guanine (G)-rich sequences, potentially forming G-quadruplex (G4) structures, are present in most replication origins in human cells. We previously suggested that the region comprising residues 413-511 of human ORC subunit 1, hORC1 413-511 , binds preferentially to G-rich DNAs, which form a G4 structure in the absence of hORC1 413-511 . Here, we investigated the interaction of hORC1 413-511 with various G-rich DNAs derived from human c-myc promoter and telomere regions. Fluorescence anisotropy revealed that hORC1 413-511 binds preferentially to DNAs that have G4 structures over ones having double-stranded structures. Importantly, circular dichroism (CD) and nuclear magnetic resonance (NMR) showed that those G-rich DNAs retain the G4 structures even after binding with hORC1 413-511 . NMR chemical shift perturbation analyses revealed that the external G-tetrad planes of the G4 structures are the primary binding sites for hORC1 413-511 . The present study suggests that human ORC1 may recognize replication origins through the G4 structure.

DNA interstrand cross-links (ICLs) are repaired in S phase by a complex, multistep mechanism involving translesion DNA polymerases. After replication forks collide with an ICL, the leading strand approaches to within one nucleotide of the ICL ("approach"), a nucleotide is inserted across from the unhooked lesion ("insertion"), and the leading strand is extended beyond the lesion ("extension"). How DNA polymerases bypass the ICL is incompletely understood. Here, we use repair of a site-specific ICL in Xenopus egg extracts to study the mechanism of lesion bypass. Deep sequencing of ICL repair products showed that the approach and extension steps are largely error-free. However, a short mutagenic tract is introduced in the vicinity of the lesion, with a maximum mutation frequency of ~1%. Our data further suggest that approach is performed by a replicative polymerase, while extension involves a complex of Rev1 and DNA polymerase ζ. Rev1-pol ζ recruitment requires the Fanconi anemia core complex but not FancI-FancD2. Our results begin to illuminate how lesion bypass is integrated with chromosomal DNA replication to limit ICL repair-associated mutagenesis., (© 2015 The Authors.)

Origin recognition complex (ORC), consisting of six subunits ORC1-6, is known to bind to replication origins and function in the initiation of DNA replication in eukaryotic cells. In contrast to the fact that Saccharomyces cerevisiae ORC recognizes the replication origin in a sequence-specific manner, metazoan ORC has not exhibited strict sequence-specificity for DNA binding. Here we report that human ORC binds preferentially to G-quadruplex (G4)-preferable G-rich RNA or single-stranded DNA (ssDNA). We mapped the G-rich RNA-binding domain in the ORC1 subunit, in a region adjacent to its ATPase domain. This domain itself has an ability to preferentially recognize G4-preferable sequences of ssDNA. Furthermore, we found, by structure modeling, that the G-rich RNA-binding domain is similar to the N-terminal portion of AdoMet_MTase domain of mammalian DNA methyltransferase 1. Therefore, in contrast with the binding to double-stranded DNA, human ORC has an apparent sequence preference with respect to its RNA/ssDNA binding. Interestingly, this specificity coincides with the common signature present in most of the human replication origins. We expect that our findings provide new insights into the regulations of function and chromatin binding of metazoan ORCs.

The facilitates chromatin transcription (FACT) complex affects nuclear DNA transactions in a chromatin context. Though the involvement of FACT in eukaryotic DNA replication has been revealed, a clear understanding of its biochemical behavior during DNA replication still remains elusive. Here, we analyzed the chromatin-binding dynamics of FACT using Xenopus egg extract cell-free system. We found that FACT has at least two distinct chromatin-binding phases: (1) a rapid chromatin-binding phase at the onset of DNA replication that did not involve origin licensing and (2) a second phase of chromatin binding that initiated after origin licensing. Intriguingly, early-binding FACT dissociated from chromatin when DNA replication was blocked by the addition of Cdc6 in the licensed state before origin firing. Cdc6-induced removal of FACT was blocked by the inhibition of origin licensing with geminin, but not by suppressing the activity of DNA polymerases, CDK, or Cdc7. Furthermore, chromatin transfer experiments revealed that impairing the later binding of FACT severely compromises DNA replication activity. Taken together, we propose that even though FACT has rapid chromatin-binding activity, the binding pattern of FACT on chromatin changes after origin licensing, which may contribute to the establishment of its functional link to the DNA replication machinery., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Mcm2-7 is recruited to eukaryotic origins of DNA replication by origin recognition complex, Cdc6 and Cdt1 thereby licensing the origins. Cdc6 is essential for origin licensing during DNA replication and is readily destabilized from chromatin after Mcm2-7 loading. Here, we show that after origin licensing, deregulation of Cdc6 suppresses DNA replication in Xenopus egg extracts without the involvement of ATM/ATR-dependent checkpoint pathways. DNA replication is arrested specifically after chromatin binding of Cdc7, but before Cdk2-dependent pathways and deregulating Cdc6 after this step does not impair activation of origin firing or elongation. Detailed analyses revealed that Cdc6 deregulation leads to strong suppression of Cdc7-mediated hyperphosphorylation of Mcm4 and subsequent chromatin loading of Cdc45, Sld5 and DNA polymerase α. Mcm2 phosphorylation is also repressed although to a lesser extent. Remarkably, Cdc6 itself does not directly inhibit Cdc7 kinase activity towards Mcm2-4-6-7 in purified systems, rather modulates Mcm2-7 phosphorylation on chromatin context. Taken together, we propose that Cdc6 on chromatin acts as a modulator of Cdc7-mediated phosphorylation of Mcm2-7, and thus destabilization of Cdc6 from chromatin after licensing is a key event ensuring proper transition to the initiation of DNA replication.

Stalled replication forks activate and are stabilized by the ATR (ataxia-telangiectasia mutated and Rad3 related)-mediated checkpoint, but ultimately, they must also recover from the arrest. Although primed single-stranded DNA (ssDNA) is sufficient for checkpoint activation, it is still unknown how this signal is generated at a stalled replication fork. Furthermore, it is not clear how recovery and fork restart occur in higher eukaryotes. Using Xenopus laevis egg extracts, we show that DNA replication continues at a stalled fork through the synthesis and elongation of new primers independent of the checkpoint. This synthesis is dependent on the activity of proliferating cell nuclear antigen, Pol-delta, and Pol-epsilon, and it contributes to the phosphorylation of Chk1. We also used defined DNA structures to show that for a fixed amount of ssDNA, increasing the number of primer-template junctions strongly enhances Chk1 phosphorylation. These results suggest that new primers are synthesized at stalled replication forks by the leading and lagging strand polymerases and that accumulation of these primers may contribute to checkpoint activation.

Purpose: To study the expected usefulness of the introduction of the DRG-PPS (Diagnosis-Related Group/Prospective Payment System, in which an insurer pays a fixed medical fee per hospitalization) into the current medical care of tuberculosis (TB) in Japan., Method: The medical fees were reviewed for all TB inpatients at 19 hospitals under the National Hospital Organization who were discharged in either June 2007 or February 2008. The sum of the fixed fee by the DRG was assumed based on the bivariate regression analysis of each patient's hospital days and his or her total actual fees during the hospital stay under the current (fee for care) system, since it was difficult to directly calculate the daily fees for every patient that would be the basis of DRG-PPS., Results: Linear regression analysis estimated that the medical fees (including fees for the medical examinations and the treatments) for a hospital stay of 60 days, which is the standard for TB treatment, was 1,192,470 yen (19,870 yen per person per day) in June 2007, and 1,167,600 yen (19,460 yen per person per day) in February 2008., Discussion: If we assume an average medical fee of about Y1.1-1.2 million yen for the standard hospital care of TB, the economic balance of the hospitals is negative, with a deficit of 0.6-0.7 million yen, given the estimated expenses of 1.8 million yen (i.e., 30,000 yen per person per day x 60 days)., Conclusion: If the DRG-PPS is to be implemented based on the current medical fee rating system, the hospital administrators could not accept its introduction to the TB medical care service as it is, because it may undermine the economic management of hospitals.

REV3 is the catalytic subunit of DNA polymerase zeta (pol zeta), which is responsible for the damage-induced mutagenesis that arises during error-prone translesion synthesis in eukaryotes. The related REV3L genes in human and mouse encode proteins of approximately 350kDa, twice as large as yeast REV3, but full-length REV3L has not been identified in any vertebrate cell. We report that Xenopus laevisREV3L encodes a 352-kDa protein that has high overall amino acid sequence similarity to its mammalian counterparts, and, for the first time in a vertebrate species, we have detected putative REV3L polypeptides of 300 and 340kDa in X. laevis oocytes. Only the 300-kDa form is stored in eggs, where its concentration of about 65pM is much lower than those of other replication and repair proteins including the accessory pol zeta subunit REV7. In fertilized eggs, the levels of this polypeptide did not change until neurula; the larger 340-kDa form first appeared at stages after gastrula, suggesting a pattern of regulation during development. These observations indicate the existence of REV3L as a scarce protein, of approximately the full predicted size, whose level may impose severe constraints on the assembly of pol zeta in X. laevis., (Copyright (c) 2009 Elsevier B.V. All rights reserved.)

When human cells enter S-phase, overlapping differential inhibitory mechanisms downregulate the replication licensing factors ORC1, CDC6 and Cdt1. Such regulation prevents re-replication so that deregulation of any individual factor alone would not be expected to induce overt re-replication. However, this has been challenged by the fact that overexpression of Cdt1 or Cdt1+CDC6 causes re-replication in some cancer cell lines. We thought it important to analyze licensing regulations in human non-cancerous cells that are resistant to Cdt1-induced re-replication and examined whether simultaneous deregulation of these licensing factors induces re-replication in two such cell lines, including human fibroblasts immortalized by telomerase. Individual overexpression of either Cdt1, ORC1 or CDC6 induced no detectable re-replication. However, with Cdt1+ORC1 or Cdt1+CDC6, some re-replication was detectable and coexpression of Cdt1+ORC1+CDC6 synergistically acted to give strong re-replication with increased mini-chromosome maintenance (MCM) loading. Coexpression of ORC1+CDC6 was without effect. These results suggest that, although Cdt1 regulation is the key step, differential regulation of multiple licensing factors ensures prevention of re-replication in normal human cells. Our findings also show for the first time the importance of ORC1 regulation for prevention of re-replication.

Object: The aim of this study was to investigate whether delayed endoscopic treatment of intraventricular hemorrhage (IVH) can prevent consecutive communicating hydrocephalus., Methods: A retrospective series of 9 patients with IVH caused by intracerebral hemorrhage (ICH) who were treated with external ventricular drainage (EVD) or endoscopic IVH removal and endoscopic third ventriculostomy (ETV) was studied in our institute. Five of these patients who had previously been treated a year before in our institute with the installation of a flexible endoscope, were treated with EVD alone on admission. Of the other patients, three received endoscopic removal of IVH and ETV and, after a one week, EVD placement, and the final patient underwent endoscopic IVH removal and ETV one day after onset., Results: Three of the patients treated with EVD alone were fitted with the EVD for 8, 11 and 16 days, and 2 patients were fitted with the EVD until they died. No patients treated with EVD alone required shunt placement. In contrast, of the 4 patients treated endoscopically, EVD was placed totally for 0, 6, 9, and 22 days for each patient, among whom 2 patients required shunt placement., Conclusions: Delayed endoscopic IVH removal and ETV might not prevent consecutive communicating hydrocephalus if IVH removal was insufficient.

In an attempt to study bone remodeling by noninvasive methods, spinal bone radiodensity was assessed in five patients treated with anterior cervical decompression and fusion (ACDF) using cylindrical titanium cages. Plain radiographs were used to study specific areas of vertebral bone interposed in two-level cages with the two cephalad vertebrae for controls. Measurements were made immediately after surgery and 1, 3, 6, 12 and 18 months postoperatively. The data were analyzed quantitatively with a contrast-comparing method (CCM) using "Scion image". There were two cyclical changes in vertebral remodeling. First, in all patients there were gradual increases in bone density at the ventral part compared to the dorsal part of the vertebral body for up to 12 months; then the density decreased at 18 months. Second, a linear gradient in radiodensity from the ventral part to the dorsal part of the vertebral body observed immediately following spinal fusion gradually disappeared by 12 months; nonhomogeneous distributions of trabecular bone were appeared. Then, the linear gradient in density appeared again at 18 months. This investigation helps elucidate the radiographic evidence for the remodeling of vertebral bone in patients treated with ACDF.

Background: DNA polymerase epsilon (Pol epsilon) is involved in DNA replication, repair, and cell-cycle checkpoint control in eukaryotic cells. Although the roles of replicative Pol alpha and Pol delta in chromosomal DNA replication are relatively well understood and well documented, the precise role of Pol epsilon in chromosomal DNA replication is not well understood., Results: This study uses a Xenopus egg extract DNA replication system to further elucidate the replicative role(s) played by Pol epsilon. Previous studies show that the initiation timing and elongation of chromosomal DNA replication are markedly impaired in Pol epsilon-depleted Xenopus egg extracts, with reduced accumulation of replicative intermediates and products. This study shows that normal replication is restored by addition of Pol epsilon holoenzyme to Pol epsilon-depleted extracts, but not by addition of polymerase-deficient forms of Pol epsilon, including polymerase point or deletion mutants or incomplete enzyme complexes. Evidence is also provided that Pol epsilon holoenzyme interacts directly with GINS, Cdc45p and Cut5p, each of which plays an important role in initiation of chromosomal DNA replication in eukaryotic cells., Conclusion: These results indicate that the DNA polymerase activity of Pol epsilon holoenzyme plays an essential role in normal chromosomal DNA replication in Xenopus egg extracts. These are the first biochemical data to show the DNA polymerase activity of Pol epsilon holoenzyme is essential for chromosomal DNA replication in higher eukaryotes, unlike in yeasts.

We describe an improved model of DNA replication in Xenopus egg extracts, in which a circular plasmid immobilized on paramagnetic beads is used as a template. DNA synthesis occurred on either circular or linear plasmids coupled to the beads, but only DNA synthesis on the circular plasmid was inhibited by geminin and a CDK inhibitor, p21. DNA synthesis on the circular plasmid occurred after a time lag, during which nuclear formation was probably occurring. Although pre-replicative complexes (pre-RCs) were formed soon after mixing plasmids with egg extracts, binding of CDC45, RPA, Pol alpha, delta and epsilon, and PCNA to the circular plasmid was delayed, but still correlated with DNA synthesis. Moreover, p21 inhibited binding of these replication fork proteins to the circular plasmid. Therefore, the circular plasmid, but not the linear plasmid, assembles bona fide replication forks in egg extracts. We conclude that this improved replication system will be useful for studying the mechanism of formation of replication forks in eukaryotic DNA replication.

Werner syndrome is a genetic disorder characterized by premature aging and cancer-prone symptoms, and is caused by mutation of the WRN gene. WRN is a member of the RecQ helicase family and is thought to function in processes implicated in DNA replication and repair to maintain genome stability; however, its precise function is still unclear. We found that replication fork arrest markedly enhances chromatin binding of focus-forming activity 1 (FFA-1), a Xenopus WRN homolog, in Xenopus egg extracts. In addition to FFA-1, DNA polymerase delta (Poldelta) and replication protein A, but not DNA polymerase epsilon and proliferating cell nuclear antigen, accumulated increasingly on replication-arrested chromatin. Elevated accumulation of these proteins was dependent on formation of pre-replicative complexes (pre-RCs). Double-strand break (DSB) formation also enhanced chromatin binding of FFA-1, but not Poldelta, independently of pre-RC formation. In contrast to FFA-1, chromatin binding of Xenopus Bloom syndrome helicase (xBLM) only slightly increased after replication arrest or DSB formation. Thus, WRN-specific, distinct processes can be reproduced in the in vitro system in egg extracts, and this system is useful for biochemical analysis of WRN functions during DNA metabolism.

We investigated the dynamics of DNA binding of replication initiation proteins during formation of the pre-replicative complex (pre-RC) on plasmids in Xenopus egg extracts. The pre-RC was efficiently formed on plasmids at 23 degrees C, with one or a few origin recognition complex (ORC) molecules and approximately 10-20 mini-chromosome maintenance 2 (MCM2) molecules loaded onto each plasmid. Although geminin inhibited MCM loading, MCM interacted weakly but stoichiometrically with the plasmid in an ORC-dependent manner, even in the presence of geminin (with approximately 10 MCM2 molecules per plasmid). Interestingly, DNA binding of ORC, CDC6, and CDT1 was significantly stabilized in the presence of geminin, under which conditions approximately 10-20 molecules each of ORC and CDC6 were bound. Moreover, a similarly stable ORC-CDC6-CDT1 complex rapidly formed on DNA at lower temperature (0 degrees C) without geminin, with approximately 10-20 molecules each of ORC and CDC6 bound to the plasmid, but almost no binding of MCM. However, upon shifting the temperature to 23 degrees C, most ORC, CDC6, and CDT1 molecules were displaced from the DNA, leaving about one ORC molecule on the plasmid, whereas approximately 10 MCM2 molecules were loaded onto each plasmid. Furthermore, it was possible to load MCM onto DNA when the isolated ORC-CDC6-CDT1-DNA complex was mixed with purified MCM proteins. These results suggest that an ORC-CDC6-CDT1 complex pre-formed on DNA is directly involved in MCM loading and imply that each DNA-bound ORC molecule loads only one or a few MCM2-7 complexes during metazoan pre-RC formation.

Background: It has been reported that combined therapy of angiotensin converting enzyme inhibitor and angiotensin receptor blocker significantly decreases proteinuria in immunoglobulin A (IgA) nephropathy. However, histologic alterations following the therapy have not been reported., Methods: A total of nine Japanese children with severe proteinuric IgA nephropathy who received a prompt immunosuppressive therapy were enrolled the study, four of whom received a combined therapy of angiotensin converting enzyme inhibitor, enalapril and angiotensin receptor blocker, losartan (Group A), while the remaining five did not (Group B). All underwent renal biopsy before and approximately 12 months after the first renal biopsy., Results: At presentation, urine protein excretion and the histologic indices of mean activity index, mean chronicity index and tubulointerstitial scores did not show a statistical difference between the two groups: Group A (2.6 +/- 0.6 g/day; mean activity index, 5.0 +/- 1.0; mean chronicity index, 5.0 +/- 1.0; tubulointerstitial scores, 4.3 +/- 1.0) and Group B (2.2 +/- 0.6 g/day; mean activity index, 4.8 +/- 0.8; mean chronicity index, 4.8 +/- 1.3; tubulointerstitial scores, 3.6 +/- 0.5, respectively). All had normal blood pressure and renal function. Urine protein excretion and the activity index decreased at the second renal biopsy, while the chronicity index and the tubulointerstitial scores slightly increased or remained unchanged. In comparison with Group B, a significant suppression in increasing the chronicity index and the tubulointerstitial scores obtained at the second renal biopsy were observed in Group A [Group A: 4.3 +/- 1.2 and 3.0 +/- 0.0, respectively, vs Group B: 6.0 +/- 0.7 and 4.4 +/- 0.9, respectively (P < 0.05)]. One patient in Group B developed chronic renal insufficiency thereafter., Conclusions: Although only a small number of patients were examined, these clinical findings suggest that a combined therapy of enalapril and losartan may attenuate histologic progression in at least a proportion of patients with severe proteinuric IgA nephropathy.

DNA polymerases delta and epsilon (Poldelta and Polepsilon) are widely thought to be the major DNA polymerases that function in elongation during DNA replication in eukaryotic cells. However, the precise roles of these polymerases are still unclear. Here we comparatively analysed DNA replication in Xenopus egg extracts in which Poldelta or Polepsilon was immunodepleted. Depletion of either polymerase resulted in a significant decrease in DNA synthesis and accumulation of short nascent DNA products, indicating an elongation defect. Moreover, Poldelta depletion caused a more severe defect in elongation, as shown by sustained accumulation of both short nascent DNA products and single-stranded DNA gaps, and also by elevated chromatin binding of replication proteins that function more frequently during lagging strand synthesis. Therefore, our data strongly suggest the possibilities that Poldelta is essential for lagging strand synthesis and that this function of Poldelta cannot be substituted for by Polepsilon.

A Japanese boy aged 14 years presented with gross hematuria associated with mild proteinuria and was diagnosed as having nutcracker syndrome. Magnetic resonance angiography (MRA) revealed significant compression of the left renal vein between the aorta and the superior mesenteric artery with collaterals. A percutaneous renal biopsy on the right kidney revealed no evidence of glomerular or interstitial changes with immune deposition. He was observed closely without any intervention thereafter. Although repeat MRA performed 4 years after our first observation disclosed the development of collateral veins, severe hematuria with an intermittent exacerbation remained unchanged. During the next 2 years, the hematuria completely subsided spontaneously. Although the etiology of spontaneous remission of the disease remains speculative, his good physical development (i.e., approximately 10 cm taller than his height at the onset) may change presumptive hemodynamic factors. These clinical observations suggest that a proportion of pubertal patients with nutcracker syndrome should be treated conservatively for a relatively long time.

Idiopathic (primary) tubulointerstitial nephritis (TIN) of childhood is relatively rare. Four children, two with concomitant uveitis, aged 8-14 years, with idiopathic TIN who underwent repeat renal biopsy were retrospectively evaluated. At presentation, all had a significant elevation of the urinary beta(2)-microglobulin/creatinine ratio (beta2MG ratio), ranging from 10100 to 44550, with increased histological indices of tubulointerstitial scores (TI scores) in excess of 6 points. Three of the children received prednisolone (PSL) therapy following diagnosis, while the remaining child received the therapy 30 months after the first renal biopsy. In the children that received prompt PSL therapy, a rapid decrease in urinary beta2MG ratio was observed and the TI scores obtained at a mean interval of 16 months after the first biopsy decreased to less than 5, while preserving renal function. In the remaining child that received delayed PSL therapy, persistent elevations of urinary beta2MG ratio and TI scores were observed. He subsequently progressed to chronic renal insufficiency. These clinical findings suggest that persistent elevations of urinary beta2MG ratio and TI scores are indicators of progression of renal failure in TIN. For successful treatment, early therapeutic intervention should be deployed in selected patients with severe idiopathic TIN.

Dbf4 is a regulatory subunit for the Cdc7 protein kinase that is required for the initiation of eukaryotic DNA replication, but the precise roles of Dbf4-Cdc7 remain to be determined. Here we identified a Xenopus homolog of Dbf4 (XDbf4) and characterized XDbf4 and Xenopus Cdc7 (XCdc7) in Xenopus egg extracts. XDbf4 formed a complex with XCdc7 in egg extracts and activated XCdc7 kinase activity in vitro. In contrast with Dbf4 in yeast and mammalian cultured cells, the XDbf4 levels in egg extracts did not change during the cell cycle progression. XDbf4 was a phosphoprotein in interphase extracts, and was apparently hyperphosphorylated in cytostatic factor (CSF)-mediated, metaphase-arrested extracts and in mitotic extracts. However, the hyperphosphorylation of XDbf4 did not seem to affect the level of kinase activation, or chromatin binding of the XDbf4-XCdc7 complex. Upon release from CSF-arrest, XDbf4 was partially dephosphorylated and bound to chromatin. Interestingly, XDbf4 was loaded onto chromatin before XCdc7 during DNA replication in egg extracts. These results suggest that the function of XDbf4-XCdc7 during the early embryonic cell cycle is regulated in a manner distinct from that during the somatic cell cycle.

A Japanese girl aged 12 years who presented with a month history of uveitis developed a significant elevation of urinary beta 2 microglobulin (beta 2MG) up to 13,933 micrograms/l. A percutaneous renal biopsy revealed a dense CD4-positive T-cell infiltration with focal tubulitis in the interstitium. The tubulointerstitial score (TI score) described by Foster et al. was 7 points. She was diagnosed as having tubulointerstitial nephritis and uveitis syndrome (TINU). Due to the severe interstitial infiltration, a 6-month course of prednisolone at the dose of 30 mg per alternate day was started. The levels of urinary beta 2MG dramatically decreased following treatment and the renal function remained normal. The second renal biopsy performed 6 months later revealed mild persistent CD4-positive T-cell infiltration associated with 19% periglomerular thickening, with the TI score of 4 points. These clinical observations suggest that the interstitial cell infiltration persists for a relatively long time in a proportion of patients with TINU. Since persistent interstitial infiltration has been known to be harmful to the kidney, we therefore speculate that prompt administration of corticosteroids might be beneficial to these patients. Although the renal outcome of TINU has been reported to be favorable to date, patients with severe interstitial infiltration should be followed under close observation. Study of similar patients is needed to clarify our understanding of effective therapy for TINU.

Nine Japanese children with severe proteinuric Henoch-Schönlein purpura nephritis (HSPN) received prompt initiation of oral prednisolone (1.5 mg/kg/day) combined with an 8-week course of cyclophosphamide (2 mg/kg/day) therapy. All underwent renal biopsy before and after treatment. At presentation, urine protein excretion and histologic indices of the mean activity index, the mean chronicity index and the tubulointerstitial (TI) scores in the patients were 5.0+/-1.4, 4.7+/-1.0, 3.9+/-1.6 and 3.7+/-0.5 g/day, respectively. Urine protein excretion, the activity index and the TI scores decreased significantly at the second renal biopsies obtained at a mean interval of 23 months after the first [0.3+/-0.3, 2.4+/-0.5 and 1.4+/-0.7 g/day ( P<0.01), respectively], while the chronicity index did not change. At the latest observation (mean interval 78 months), all except two showed negative proteinuria while no patient showed renal impairment. Although this case series is without controls, our experience suggests that early treatment with oral prednisolone and cyclophosphamide may be beneficial to a proportion of patients with severe proteinuric HSPN.

A Japanese male infant presented with nephrotic syndrome at 41 days. His renal function progressively deteriorated, and he died at 4 months of the age. An open renal biopsy revealed diffuse crescentic glomerulonephritis (CrGN) without immune complex deposition, which is not characteristic of the congenital nephrotic syndrome (CNS). Examination for nephrin antigen using rabbit anti-nephrin extra- and intracellular site antibodies was positive. These clinical observations suggest that the patient had a unique histological variant of CNS. This is the first report of rapidly progressive, pauci-immune diffuse CrGN in infancy.

Two Japanese female adolescents with systemic lupus erythematosus (SLE), known cases of urinary tract involvements: one with biopsy-proven class II lupus nephritis and the other one with lupus cystitis without overt glomerulonephritis (silent lupus), who after more than 4 years' observation presented with subsequent progression to membranous glomerulonephritis (MGN) following exacerbation of urticarial rash. Although it is well known that lupus nephritis shows histological transformation with time, the late progression to MGN from another World Health Organization histologic pattern has been reported to be less common in pediatric-onset SLE. Although pathogenesis of their MGN remains speculative, these clinical observation might suggest that a possible association between exacerbation of urticarial rash and subsequent progression to MGN in the selected patients with SLE.

A Japanese girl aged 13 years with myeloperoxidase anti-neutrophil cytoplasmic antibodies(MPO-ANCA)-associated glomerulonephritis(GN) progressed to end-stage renal failure after 7 years' clinical observation. She had been suffering from recurrent disease flare associated with serum MPO-ANCA elevation(i.e. 153 EU/ml, 208 EU/ml and 358 EU/ml, maximum at each of the episodes, normal < 10 EU/ml). Each flare was treated successfully with prednisolone combined with cyclophosphamide and azathioprine. However, her renal function gradually deteriorated, and peritoneal dialysis(PD) was initiated 7 years after the onset of the disease. During the clinical course, no extrarenal manifestations were observed. Due to subsidence of the serum MPO-ANCA titer(10 EU/ml) after starting PD, prednisolone and azathioprine were tapered thereafter. Her daily urine volume was preserved at approximately 600 ml at that time. She suddenly developed fatigue with severe anemia, oliguria and hypertension 4 months after discontinuation of immunosuppressive therapy. The serum titer of MPO-ANCA increased to 100 EU/ml. These clinical observation suggests that disease flare may occur in selected patients with MPO-ANCA-associated GN, who develop end-stage renal failure requiring PD. Although recurrent flare associated with an increased serological activity in a proportion of patients with lupus nephritis who have received dialysis has been reported to date, to our knowledge, a similar clinical observation in the MPO-ANCA-associated GN has not been reported. Selected patients with the disease should be followed with close observation after undergoing dialysis.

A Japanese girl aged 8 years who presented with a 2-month history of uveitis subsequently developed tubulointerstitial nephritis. A percutaneous renal biopsy revealed massive interstitial mononuclear cell infiltrates consisting of CD4-positive T cells. Despite administration of topical corticosteroids, the ocular symptoms persisted. Systemic corticosteroid therapy dramatically reduced the ocular symptoms and urinary beta2-microglobulin (beta 2MG) concentration. However, reducing the prednisolone dosage induced recurrence of uveitis associated with increased levels of urinary beta 2MG. The CD4-positive T cell infiltration persisted in the second renal biopsy performed 6 months after the first renal biopsy. These observations suggest that the interstitial cell infiltration persists for a relatively long time in a proportion of patients with tubulointerstitial nephritis and uveitis syndrome (TINU). Although the renal outcome of TINU has been reported to be favorable, prolonged interstitial cell infiltration may affect long-term renal outcome. Selected patients with TINU should be followed with close observation.