Crystal structure of the motor domain of centromere-associated protein E in complex with a non-hydrolysable ATP analogue.

Centromere-associated protein E (CENP-E) is a kinesin motor protein essential for mitosis and a new target for anticancer agents with less side effects. To rationally design anticancer drug candidates based on structure, it is important to determine the three-dimensional structure of the CENP-E motor domain bound to its inhibitor. Here, we report the first crystal structure of the CENP-E motor domain in complex with a non-hydrolysable ATP analogue, adenylyl-imidodiphosphate (AMPPNP). Furthermore, the structure is compared with the ADP-bound form of the CENP-E motor domain as well as the AMPPNP-bound forms of other kinesins. This study indicates that helix α4 of CENP-E participates in the slow binding of CENP-E to microtubules. These results will contribute to the development of anticancer drugs targeting CENP-E.

Crystal structures of human glyoxalase I and its complex with TLSC702 reveal inhibitor binding mode and substrate preference.

Human glyoxalase I (hGLO I) is an enzyme for detoxification of methylglyoxal (MG) and has been considered an attractive target for the development of new anticancer drugs. In our previous report, the GLO I inhibitor TLSC702 induced apoptosis in tumor cells. Here, we determined the crystal structures of hGLO I and its complex with TLSC702. In the complex, the carboxyl O atom of TLSC702 is coordinated to Zn2+ , and TLSC702 mainly shows van der Waals interaction with hydrophobic residues. In the inhibitor-unbound structure, glycerol, which has similar functional groups to MG, was bound to Zn2+ , indicating that GLO I can easily bind to MG. This study provides a structural basis to develop better anticancer drugs.

Structure and comparison of the motor domain of centromere-associated protein E.

Centromere-associated protein E (CENP-E) plays an essential role in mitosis and is a target candidate for anticancer drugs. However, it is difficult to design small-molecule inhibitors of CENP-E kinesin motor ATPase activity owing to a lack of structural information on the CENP-E motor domain in complex with its inhibitors. Here, the CENP-E motor domain was crystallized in the presence of an ATP-competitive inhibitor and the crystal structure was determined at 1.9 Šresolution. In the determined structure, ADP was observed instead of the inhibitor in the nucleotide-binding site, even though no ADP was added during protein preparation. Structural comparison with the structures of previously reported CENP-E and those of other kinesins indicates that the determined structure is nearly identical except for several loop regions. However, the retention of ADP in the nucleotide-binding site of the structure strengthens the biochemical view that the release of ADP is a rate-limiting step in the ATPase cycle of CENP-E. These results will contribute to the development of anticancer drugs targeting CENP-E and to understanding the function of kinesin motor domains.

[Adult male with chronic renal failure due to reflux nephropathy that was possibly induced by neurogenic bladder since childhood].

A 38-year-old male with impaired renal function and serious anemia was admitted to our hospital. He had suffered from a disorder of urination since early childhood and been diagnosed as having neurogenic bladder by an urologist when he was 20 years old. Since February 2007, general fatigue emerged and gradually worsened. In addition, he began to feel nauseous around February 2008. He visited a family doctor and was diagnosed with renal failure and anemia both of which were serious. H e was referred t o our office and admitted immediately because his blood test showed a serum creatinine level of 4.4 mg/dL and hemoglobin of 3.1 g/dL. The initial study with ultrasonograhy suggested that both kidneys contained multiple cysts for the most part, likely due to hereditary polycystic kidney disease. However, a subsequent series of diagnostic imaging tests, including computed tomography and magnetic resonance urography, determined that the cause of renal failure was most likely reflux nephropathy due to secondary vesicouretral reflux induced by the chronic neurogenic bladder. Moreover, the upper gastrointestinal endoscopic examination showed that the cause of anemia was probably the persistent bleeding from gastric antral vascular ectasia. Reflux nephropathy emerges in early childhood and slowly progresses to chronic renal failure in some cases. According to the literature, it is not rare as a cause of end-stage kidney failure even among adult populations. Diagnostic imaging of severe reflux nephropathy is apparently similar to that of polycystic kidney disease. We herein present an adult male with chronic renal failure due to reflux nephropathy, the images of which were similar to polycystic kidney disease.

[Recurrence of severe hypophosphatemia associated with tumor neogenesis in a patient with acute lymphocytic leukemia who experienced acute renal failure due to tumor lysis syndrome].

We report a 47-year-old male patient with acute lymphocytic leukemia (ALL) who showed recurrent severe hypophosphatemia. Chemotherapy for ALL induced tumor lysis syndrome requiring hemodialysis therapy. Thereafter, severe hypophosphatemia (serum phosphorus concentration less than 0.7 mg/dL) was observed several days before an acute rise in peripheral lymphoblast cell counts due to recurrence of ALL. This hypophosphatemia soon returned to a higher than normal level by treating leukemic cells with anti-cancer agents. This event occurred twice in his clinical course. The laboratory tests strongly suggested that this hypophosphatemia was induced by a shift of phosphorus into leukemic cells that rapidly replicated in the tissues.

[Prevalence of chronic kidney disease (CKD) and significant contributors to CKD in HIV-infected patients].

We investigated the prevalence of CKD and factors associated with CKD in HIV-infected patients who were under stable medical control. We retrospectively abstracted the medical records of 748 HIV-infected outpatients(659 males and 89 females). Their mean age was 44.9+/-11.7 (range; 21 to 79) years. The following parameters were reviewed: urinalysis including proteinuria and microscopic hematuria, urinary N-acetyl-beta-glucosaminidase (NAG) level as a marker of tubular damage, serum creatinine level, estimated glomerular filtration rate calculated based on the Modification of Diet in Renal Disease formula (eGFR), CD4 count in peripheral blood, HIV-RNA copies in serum, use and vintage of highly active antiretroviral therapy (HAART), and use of anti-hypertensive drugs (AHTD). Stages of CKD were determined based on the K/DOQI stages of kidney disease. Chronic renal failure (CRF) was defined as an eGFR value of less than 60 mL/min/1.73 m2. The chi-square test was used to evaluate differences in the prevalence of dichotomous variables. Multivariable logistic regression analysis was applied to assess independent contributors to existence of CRF, proteinuria, and tubular damage. Proteinuria was positive in 50.0 % and hematuria was positive in 11.3 %. Both were positive in 8.41%. Tubular damage (> 11 U/L of urinary NAG levels) was positive in 42 out of 112(37.5 %). Prevalence of CKD and CRF was 87.8 % and 16.2 %, respectively. Stages of CKD were: stage 5D, 4 patients (0.53 %); stage 5, 0 patients (0%); stage 4, 3 patients (0.40 %); stage 3, 114 patients (15.2 %); stage 2, 487 patients (65.1%); stage 1, 49 patients (6.6%); and non-CKD, 91 patients (12.2 %). Statistically, use of HAART, urinary NAG level, and age were significant contributors to proteinuria. Proteinuria, age, and use of AHTD were strong predictors for CRF. Tubular damage was related to HAART vintage, age, and TG levels. In addition, HAART vintage of more than 2.5 years was statistically associated with the existence of tubular damage in HIV-infected patients. Prevalence of CKD in stable HIV-infected patients was unexpectedly high in our hospital. Aged patients with a long HAART vintage who have proteinuria and hypertension are predisposed to the development of CRF through tubular damage.

[Chronic renal failure in patients successfully treated with hematopoietic cell transplantation].

Recent development of hematopoietic cell transplantation (HCT) has greatly improved the quality of life in critical patients with hematological malignancies. On the other hand, it is a fact that some HCT survivors suffer from chronic renal failure (CRF). We attempted to examine the clinical characteristics of CRF in patients who were successfully treated with HCT in Japan. A retrospective analysis of 158 long-term survivors receiving HCT at Komagome Hospital was undertaken. CRF was designated as less than 30 mL/min of estimated GFR (eGFR) calculated by the MDRD formula. We statistically analyzed the influences of total body irradiation (TBI), graft versus host diseases (GVHD), renal impairment during HCT, new incidence of hypertension after transplantation, age, and gender on CRF, using the multivariate logistic regression analysis. Twenty-seven patients (17.1%) had CRF. Their mean ages were 33.1 +/- 8.87 years and mean eGFR levels were 20.5 +/- 9.50 mL/min/1.73 m2. Fifteen patients were recipients of TBI (55.6 %). CRF became obvious within one year after BMT in 5 patients (18.5%) and later in 22 patients (81.5%). Seven patients(25.9%) finally reached end-stage renal disease (ESRD) at the time of over 10 years after HCT. Multivariate logistic regression analysis showed that TBI, renal impairment during HCT, and new incidence of hypertension after HCT were significantly associated with CRF. Considering that 12 patients without TBI (44.4%) developed CRF, "renal impairment during HCT", the odds ratio of which was the highest, might be the factor most closely associated with CRF. The clinical course of a representative patient who developed ESRD was described. An increase in ESRD patients receiving HCT should be anticipated and would constitute a new important issue in nephrology.

[Nephrotic syndrome in patients after successful myeloablative allogeneic hematopoietic stem cell transplantation: clinical findings obtained from four transplanted patients].

We experienced four patients who suffered from nephrotic syndrome after a successful allogeneic hematopoietic stem cell transplantation (HSCT). These cases were seen in the nineteen-year period from September, 1986 to June, 2005. Our data showed that the incidence of nephrotic syndrome was 0.51% (3 out of 585 HSCT patients) in our hospital. Pathological findings of their renal biopsy specimens revealed that 3 patients had membranous nephropathy and that one patient had minimal change disease. Three patients were positive for anti nuclear antibody. Administration of prednisolone or cyclosporine improved the nephrotic syndrome, leading all patients to a complete or almost complete remission. The nephrotic syndrome occurred at 17 to 25 months after HSCT and accompanied the relapse of chronic graft-versus-host disease (GVHD), possibly due to the termination or a decrease of immunosuppressant administration in all patients. This suggests that immunological abnormality associated with chronic GVHD may be partly involved.

Reduced capacity of mononuclear cells to synthesize cytokines against an inflammatory stimulus in uremic patients.

BACKGROUND: The local production of cytokines and phagocytosis is pivotal in innate immunity. Uremic patients have a high infectious morbidity, but it remains unclear if this arises from incompetence of these local cellular functions. METHODS: In 30 predialysis uremic patients and 20 controls, we studied the intracellular cytokine synthesis by mononuclear cells in response to stimulation. Moreover, phagocytic activity by leukocytes was tested. Lipopolysaccharide- or mitogen-stimulated peripheral blood cells were labeled with anti-CD14 and -CD4 antibodies, respectively and subjected to intracellular cytokine staining and flow cytometry. Tumor necrosis factor (TNF)-alpha, IL-1beta, IL-6, and IL-8 synthesis was examined in CD14(+) monocytes. IFN-gamma and IL-4 synthesis was examined in CD4(+) helper T cells to determine their Th1 or Th2 phenotype. The flow cytometric analysis of phagocytosis of opsonized bacteria was performed in whole blood. RESULTS: Uremic patients exhibited a significantly reduced monokine response and inhibited development of helper T cells into Th1 or Th2 phenotypes compared with control subjects. Their phagocytic activity was comparable to control subjects. No clinical parameters were linked to in vitro cytokine production and phagocytic activity. CONCLUSIONS: Mononuclear cells in uremic patients are hyporeactive to inflammatory challenge and this may be one reason why uremic patients are vulnerable to infections.

Impairment of innate cellular response to in vitro stimuli in patients on continuous ambulatory peritoneal dialysis.

BACKGROUND: Most crucial in the initial stages of host defence against invading micro-organisms is innate immunity, in which peripheral mononuclear cells, in particular cytokines, are pivotal. Mortality from infections is high in dialysis patients, but it remains unclear if this arises from the ineffectiveness of innate immune mechanisms. METHODS: In 20 haemodialysis (HD) patients, 20 patients on continuous ambulatory peritoneal dialysis (CAPD), and 15 age-matched controls, we studied cytokine production by monocytes and helper T-cells in response to in vitro stimuli. The most important early-response cytokines for innate immunity, tumour necrosis factor (TNF)-alpha and interleukin (IL)-1beta, were tested in monocytes, and interferon-gamma and IL-4 were studied as indicators of polarization of helper T-cells into type 1 and type 2 cells. Peripheral blood cells stimulated with lipopolysaccharide or mitogen were labelled with anti-CD14+ and -CD4+ antibodies and then subjected to intracellular cytokine staining and flow cytometry. RESULTS: CAPD patients showed significantly reduced synthesis of TNF-alpha and IL-1beta and inhibited T helper phenotype development compared with HD patients and control subjects. In contrast, HD patients showed an unaltered monokine response and a marked polarization of helper T-cells towards the type 1 phenotype. We also found that a single HD treatment potentiated monocytes to synthesize TNF-alpha. CONCLUSIONS: Circulating immune cells in CAPD patients may be hyporeactive against infections, indicating an unfavourable innate host defence.

[Innate immunity in hemodialysis and continuous ambulatory peritoneal dialysis patients: impaired cytokine synthetic response to ex-vivo stimuli in mononuclear cells].

Dialysis patients are weak in immune host defense, which is associated with their high morbidity of infection. Polymorphonuclear leukocytes (PMNLs) and mononuclear cells play a key role in innate host defense. PMNLs and monocytes have bactericidal activity through the process of phagocytosis. Monocytes and lymphocytes contribute to the development of innate immunity by their cytokine actions. We studied the intracellular cytokine syntheses in response to ex-vivo stimuli, which may reflect the potential reactivity of immune cells in cytokine syntheses when pathogens invade humans. Furthermore, phagocytic activity was assessed in granulocytes and monocytes. Twenty HD, 15 CAPD, and 10 age-matched controls were enrolled in this study. One milliliter of whole blood from each subject was incubated with lipopolysaccharides (LPS) or mitogens for 4 hours at 37 degrees C. Monoclonal antibodies to CD14+ and CD4+ were used for identifying monocytes and helper T cells, respectively. Intracellular cytokines were stained using FASTIMMUNE staining kits. Interleukin-1beta and TNF-alpha syntheses were examined in monocytes, which are the most important early-response cytokines in innate immunity. IFN-gamma and IL-4 syntheses were examined in helper T cells to observe their polarization into Th1 and Th2 cells. IFN-gamma is a key factor in establishing innate immunity. The percentage of cells that stained positive for each cytokine was analyzed using a flow cytometer. The following results were obtained: 1) In CAPD patients, IL-1beta and TNF-alpha response to LPS in monocytes were significantly reduced, as compared to other subjects. Polarization of helper T cells was reduced, resulting in a significant decrease in Th1 cells. 2) In HD patients, monokine responses were not altered, but polarization of helper T cells was skewed toward a Th1 type. Phagocytic activities were not impaired in both dialysis groups. In conclusion, mononuclear cells from CAPD patients have the potential to exhibit failure of a cytokine response to ex-vivo stimuli in terms of innate immunity.