oriyama M

Induction of centrosome amplification and chromosome instability in human bladder cancer cells by p53 mutation and cyclin E overexpression

Centrosome amplification frequently occurs in human cancers and is a major cause of chromosome instability (CIN). In mouse cells, centrosome amplification can be readily induced by loss or mutational inactivation of p53. In human cells, however, silencing of endogenous p53 alone does not induce centrosome amplification or CIN, although high degrees of correlation between p53 mutation and CIN/centrosome amplification in human cancer can be detected, suggesting the presence of additional regulatory mechanism(s) in human cells that ensures the numeral integrity of centrosomes and genomic integrity. Cyclin E, a regulatory subunit for CDK2 that plays a key role in centrosome duplication, frequently is overexpressed in human cancers. We found that cyclin E overexpression, together with loss of p53, efficiently induces centrosome amplification and CIN in human bladder cancer cells but not by either cyclin E overexpression or loss of p53 alone. We extended these findings to bladder cancer specimens and found that centrosome amplification is strongly correlated with concomitant occurrence of cyclin E overexpression and p53 inactivation but not with either cyclin E overexpression or p53 inactivation alone. Because cyclin E expression is strictly controlled in human cells compared with mouse cells, our findings suggest that this stringent regulation of cyclin E expression plays an additional role underlying numeral homeostasis of centrosomes in human cells and that deregulation of cyclin E expression, together with inactivation of p53, results in centrosome amplification.

Isolation and partial characterization of crystal matrix protein as a potent inhibitor of calcium oxalate crystal aggregation : Evidence of activation peptide of human prothrombin

In order to clarify the characteristics of crystal matrix protein (CMP), which exhibits a remarkable affinity for calcium oxalate crystals and may be important in stone pathogenesis, we have isolated CMP from macromolecular matrix substances of newly-formed calcium oxalate crystals. Purification of CMP consisted of calcium oxalate crystal formation, dissolution of crystals, electrodialysis, anion exchange chromatography and high-performance liquid chromatography. CMP showed the protein band of 31 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of CMP was identical to that of human prothrombin. Both anti-CMP polyclonal antibody and antihuman prothrombin antibody cross-reacted well with human prothrombin and CMP in Western blotting. Its amino acid composition and its molecular weight of 31 kDa strongly suggest that CMP is the activation peptide of human prothrombin.

Centrosome Hyperamplification and Chromosomal Instability in Bladder Cancer

OBJECTIVE Chromosomal instability (CIN) is a common feature of malignant tumors. Centrosome hyperamplification (CH) occurs frequently in human cancers, and may be a contributing factor in CIN. In this study, we investigated the relationship between CH and CIN in bladder cancer. METHODS Clinical samples obtained by transurethral resection from 22 patients with bladder cancer were examined (histological grade G1, 5 cases; G2, 6 cases; G3, 11 cases). CH was evaluated by immunohistochemistry using anti-pericentrin antibody. CIN was evaluated by fluorescence in situ hybridization (FISH). FISH probes for pericentromeric regions of chromosomes 3, 7, and 17 were hybridized to touch preparations of nuclei from frozen tissues. We also analyzed the centrosome replication cycle of bladder cancer by laser scanning cytometry (LSC). RESULTS Of the 22 cases examined, 18 (81.8%) had centrosome hyperamplification: CH 0, 4 cases (18.1%); CH I, 5 cases (22.7%); CH II, 5 cases (22.7%); CH III, 8 cases (36.4%). The grade of CH was directly proportional to the histological grade (p=0.03, chi(2) test). LSC analysis showed that the centrosome replication cycle was well regulated in pathologically low-grade bladder cancer, which did not have chromosomal instability. In contrast, we found marked variability of centrosomes in pathologically high-grade bladder cancer, which had chromosomal instability. CH and CIN were both detected in pathologically high-grade tumors. The grade of CH was directly proportional to the CIN grade (p=0.0079, chi(2) test). CONCLUSION The results of the present study suggest that CH may be involved in CIN in bladder cancer.

Inhibitions of urinary oxidative stress and renal calcium level by an extract of Quercus salicina Blume/Quercus stenophylla Makino in a rat calcium oxalate urolithiasis model.

Objectives:  To clarify the mechanism of Urocalun, an extract of Quercus salicina Blume/Quercus stenophylla Makino (QS), in the treatment of urolithiasis.

Renal protective effects of erythropoietin on ischemic reperfusion injury.

While the problem of organ shortage has not yet been solved, the number of patients who need to be treated with dialysis due to end-stage renal disease (ESRD) is increasing each year. With the aim of eliminating dialytic therapy as much as possible, the opportunities for organ donation from expansive criteria donor (ECD) or marginal donors due to cardiac death have been increasing. With the purpose of extracting organs in a state in which the function is preserved as much as possible, we reexamined the conditions of tissue disorders resulting from temporary ischemia of the organs as well as changes in tissue function and the effects on the preservation of renal function over time by using rat models in order to clinically utilize erythropoietin, which has inhibitory effects on ischemia-reperfusion disorder, as has been conventionally reported. With 8- to 9-week-old Wister male rats, after the right kidney had been resected under general anesthesia, the left renal artery was clamped to inhibit the blood flow for 45 min. At 30 min before inhibiting the blood flow and after releasing the inhibited blood flow, 100 U/kg of recombinant human erythropoietin (rhEPO) was administered via the inferior vena cava and the abdominal cavity, and then the tissues and blood samples were extracted at 6 and 24 h after the release. The renal tissue specimens were evaluated using H&E staining and TUNEL staining in order to observe differences in the expression of apoptosis as well as the renal function and changes in the emergence of active oxygen were investigated by using samples that had been obtained from drawn blood. Moreover, we examined the degree of renal dysfunction by means of neutrophil gelatinase-associated lipocalin (NGAL) in the spot urine samples. The changes in renal function, which were observed according to the serum creatinine level, showed that the renal function was preserved with a significant difference in the rhEPO administration group. The liver deviation enzymes, which had also shown increases in the serum as well as the occurrence of renal dysfunction, showed clear decreases in the serum, even though changes with a significant difference were not observed in the rhEPO administration group. The active oxygen did not show changes before and after ischemia-reperfusion nor changes due to the rhEPO administration. When examining the status of apoptosis in the tissues, apoptosis was shown to be inhibited due to the rhEPO administration. It is believed that the main preservation effects of rhEPO are the elimination of cytopathy/cell death, as derived from the resulting ischemic condition that extends to the target organ before ischemia occurs. In this examination, no direct effects of rhEPO administration on the emergence of active oxygen were observed. It is therefore suggested that there is a possibility of preserving the renal function in marginal donors with a longer agonal stage by effectively using rhEPO.

BACKSCATTERED ELECTRON IMAGING OF CRYSTAL MATRIX PROTEIN ON THE SURFACE OF CALCIUM OXALATE CRYSTALS USING COLLOIDAL GOLD

In order to clarify the presence and localization of crystal matrix protein (CMP) upon calcium oxalate crystals, scanning electron microscopy (SEM) and backscattered electron imaging (BEI) techniques were used. This protein exhibits a remarkable affinity with calcium oxalate crystals and may be important in stone pathogenesis. In this paper, rabbit anti‐human CMP polyclonal antibody was used as first antibody, and for the second antibody, goat anti‐rabbit IgG conjugated with 20 nm immunogold was used. Freshly prepared crystals from male urine were fixed in SEM fixative, then blocked and washed with phosphate‐buffered saline and bovine serum albumin (PBS/BSA). First and second antibodies were reacted in PBS/BSA. Crystals were then dehydrated and finally coated for SEM study. The SEM technique showed bipyramidal shaped dihydrate calcium oxalate crystals in every sample and even at high magnification, colloidal gold could barely be seen. BE 1 clearly demonstrated the presence and localization of the gold on the surface of the crystals as well as on the macromol‐ecules eluted from the crystals by dissolving them in ethylenediamminetetraacetic acid solution.

A case of second renal transplantation with acute antibody-mediated rejection complicated with BK virus nephropathy

Atsumi H, Asaka M, Kimura S, Imura J, Fujimoto K, Chikazawa Y, Nakagawa M, Okuyama H, Yamaya H, Moriyama M, Tanaka T, Suzuki K, Yokoyama H. A case of second renal transplantation with acute antibody‐mediated rejection complicated with BK virus nephropathy.
Clin Transplant 2010: 24 (Suppl. 22): 35–38.

Proteomic analysis of a rare urinary stone composed of calcium carbonate and calcium oxalate dihydrate: A case report

The objective of the present study was to investigate the matrix protein of a rare urinary stone that contained calcium carbonate. A urinary stone was extracted from a 34‐year‐old male patient with metabolic alkalosis. After X‐ray diffractometry and infrared analysis of the stone, proteomic analysis was carried out. The resulting mass spectra were evaluated with protein search software, and matrix proteins were identified. X‐ray diffraction and infrared analysis confirmed that the stone contained calcium carbonate and calcium oxalate dihydrate. Of the identified 53 proteins, 24 have not been previously reported from calcium oxalate‐ or calcium phosphate‐containing stones. The protease inhibitors and several proteins related to cell adhesion or the cytoskeleton were identified for the first time. We analyzed in detail a rare urinary stone composed of calcium carbonate and calcium oxalate dihydrate. Considering the formation of a calcium carbonate stone, the new identified proteins should play an important role on the urolithiasis process in alkaline condition.

Catheter blockage factors in patients cared for in their own home requiring long‐term urinary catheterisation

Early urinary catheter removal from patients cared for in their own home is often difficult because of incontinence and care burdens, resulting in long duration of catheterization. Urinary tract infection and catheter blockage are major complications associated with long-term catheterization. Preventing these complications is important for patients who require long-term catheterization in their own home. This study aimed to examine the occurrence characteristics of urinary catheter blockage and to identify the causative factors of catheter blockage in patients cared for in their own home requiring long-term urinary catheterization. The study targeted 154 patients cared for in their own home requiring long-term urinary catheterization. Patient data were collected by questionnaire by the nurse manager. Catheter blockage occurred in 33·8% of patients requiring long-term catheterization within the first 6 months, and the mean frequency in these patients was 3·0, indicating that catheter blockage occurs repeatedly. The following management factors significantly correlated with the frequency of catheter blockage: ‘check urine volume’, ‘check for flexion and distortion of the catheter’ and ‘check urinary tract infection symptoms’ for catheter management by caregivers. Symptoms correlated with catheter blockage included ‘cloudiness of the urine’, ‘decreased urine volume’, ‘abdominal pain’ and ‘duration of catheterization’. In the future, we would like to prepare the protocol to help visiting nurses improve their abilities to manage catheter blockage in patients requiring long-term catheterization, by incorporating the findings from this study.