Prediction of Early BK Virus Infection in Kidney Transplant Recipients by the Number of Cells With Intranuclear Inclusion Bodies (Decoy Cells).

BACKGROUND: BK virus (BKV) is the cause of nephropathy. Because BKV nephropathy can progress to graft loss, early diagnosis of BKV infection is very important. In this study, we aimed to investigate the utility of quantifying cells with intranuclear inclusion bodies (decoy cells) in urinary sediment for the screening and monitoring of BKV infection in renal transplant recipients at our hospital. METHODS: This was a retrospective single-center study. Urine sediment examination was performed at each outpatient visit, and the number of decoy cells was measured in the whole microscopic field. Patients (n = 41) were divided into the BK viremia group (blood positive for BKV DNA by polymerase chain reaction [PCR]) and non-BK viremia group (blood negative for BKV DNA by PCR), and the decoy cell count in urinary sediments was examined. RESULTS: The maximum decoy cell count was significantly higher (P = 0.04) in the BK viremia group than in the non-BK viremia group. In the receiver operating characteristic curve for the maximum decoy cells, the cutoff value was 507 cells. The area under the receiver operating characteristic curve was 0.8774 (95% confidence interval, 0.7739-0.9810). The number of decoy cells at the time of appearance in the BK viremia group was not significantly different from that in the non-BK viremia group. However, the BK viremia group showed an increasing trend, whereas the non-BK viremia group showed a decreasing trend, in the number of decoy cells. There was a positive correlation between the number of decoy cells and the data from the urine BKV-DNA PCR quantification (correlation coefficient [r] = 0.74). CONCLUSIONS: Measurement of decoy cells in urinary sediments may predict early BKV infection, and if performed quickly, it may be useful for screening and continuous monitoring of BKV infection in renal transplant recipients.

Emergence of Mycoplasma genitalium with clinically significant fluoroquinolone resistance conferred by amino acid changes both in GyrA and ParC in Japan.

We observed fluoroquinolone treatment failures in 2 men with Mycoplasma genitalium-positive non-gonococcal urethritis in Japan. A fluoroquinolone regimen of sitafloxacin 100 mg twice daily for 7 days failed to eradicate M. genitalium. In both cases, M. genitalium had fluoroquinolone resistance-associated amino acid changes both in GyrA and ParC and a macrolide resistance-associated mutation in the 23S rRNA gene. The emergence of such multi-drug resistant strains can threaten antimicrobial chemotherapy for M. genitalium infections in Japan, because we will lose the first- (azithromycin) and second-line (sitafloxacin) antimicrobial agents to treat M. genitalium infections. We prescribed an extended minocycline regimen of minocycline 100 mg twice daily for 14 days for our patients, and the regimen was successful in eradicating the M. genitalium. The extended minocycline regimen might be an option that we can try when treating multi-drug resistant M. genitalium infections in clinical practice.

Clinical courses of herpes simplex virus-induced urethritis in men.

We retrieved clinical data of 13 men having herpes simplex virus (HSV)-induced non-gonococcal urethritis (NGU) without visible herpetic lesions. They visited a clinic in Sendai, Japan, between April 2013 and December 2015. All the men complained of dysuria. Meatitis was observed in 9 of the 13 men. Mononuclear cells were observed in the urethral smears from 9 men. The 13 men were treated with azithromycin or sitafloxacin regimen. First-voided urine (FVU) specimens became negative for HSV in 8 of the 10 men who returned to the clinic after antibacterial treatment, and urethritis symptoms were alleviated. However, herpetic lesions were observed at the follow-up visits in 3 men, and 2 of them were still positive for HSV in their FVU. HSV could be a cause of acute urethritis without causing visible herpetic lesions. The shedding of HSV from the urethra would spontaneously cease with alleviation of urethritis symptoms in most cases of HSV-induced NGU without antiviral therapy. However, new herpetic lesions could be developed in some cases. Early antiviral therapy is beneficial for patients with HSV infections. The development of meatitis and the mononuclear cell response in the urethral smear could be helpful to diagnose HSV-induced NGU. Therefore, we should presumptively initiate anti-HSV therapy for patients with signs and symptoms suggestive of HSV-induced NGU at their first presentation.

Ferritin Assembly Revisited: A Time-Resolved Small-Angle X-ray Scattering Study.

The assembly reaction of Escherichia coli ferritin A (EcFtnA) was studied using time-resolved small-angle X-ray scattering (TR-SAXS). EcFtnA forms a cagelike structure that consists of 24 identical subunits and dissociates into dimers at acidic pH. The dimer maintains nativelike secondary and tertiary structures and is able to reassemble into a 24-mer when the pH is increased. The reassembly reaction was induced by pH jump, and reassembly was followed by TR-SAXS. Time-dependent changes in the forward scattering intensity and in the gyration radius suggested the existence of a significant population of intermediate oligomers during the assembly reaction. The initial reaction was a mixture of second- and third-order reactions (formation of tetramers and hexamers) from the protein concentration dependence of the initial velocity. The time-dependent change in the SAXS profile was roughly explained by a simple model in which only tetramers, hexamers, and dodecamers were considered as intermediates.

Relationship between chain collapse and secondary structure formation in a partially folded protein.

Chain collapse and secondary structure formation are frequently observed during the early stages of protein folding. Is the chain collapse brought about by interactions between secondary structure units or is it due to polymer behavior in a poor solvent (coil-globule transition)? To answer this question, we measured small-angle X-ray scattering for a series of ß-lactoglobulin mutants under conditions in which they assume a partially folded state analogous to the folding intermediates. Mutants that were designed to disrupt the secondary structure units showed the gyration radii similar to that of the wild type protein, indicating that chain collapse is due to coil-globule transitions.

Clinical effectiveness and safety of tazobactam/piperacillin 4.5 g for the prevention of febrile infectious complication after prostate biopsy.

We investigated the clinical effectiveness and safety of tazobactam/piperacillin (TAZ/PIPC) in a 1:8 ratio, a ß-lactamase inhibitor with penicillin antibiotic, for the prevention of febrile infectious complication after prostate biopsy. Each patient received a single dose of TAZ/PIPC 4.5 g, 30 min before the biopsy in Group 1 or TAZ/PIPC 4.5 g twice, once 30 min before and once after the biopsy (just before discharge or 5 h after the biopsy), in Group 2. Estimation of efficacy was performed within 1-month after prostate biopsy. Clinical diagnosis of febrile infectious complication was based on a body temperature elevation greater than 38 °C. Infectious complication after prostate biopsy was detected in 2.5% (4/160 patients) in Group 1 and in 0.45% (2/442 patients) in Group 2. All of the patients with febrile infectious complication had risk factors: 5 patients had voiding disturbance, 2 patients had diabetes mellitus and 1 patient had steroid dosing. In group 1, 88 patients had at least one risk factor and 72 patients had no risk factors. Of the patients with a risk factor, 4 had febrile infectious complication after prostate biopsy, but there was no significant difference between the two groups. In group 2, 87 patients had at least one risk factor and 255 patients had no risk factors. The patients with a risk factor had febrile infectious complication significantly more frequently than did patients without a risk factor (P = 0.038). Therefore, TAZ/PIPC appears to be effective as preoperative prophylaxis against the occurrence of febrile infectious complication after prostate biopsy.

A compact intermediate state of calmodulin in the process of target binding.

Calmodulin undergoes characteristic conformational changes by binding Ca(2+), which allows it to bind to more than 300 target proteins and regulate numerous intracellular processes in all eukaryotic cells. We measured the conformational changes of calmodulin upon Ca(2+) and mastoparan binding using the time-resolved small-angle X-ray scattering technique combined with flash photolysis of caged calcium. This measurement system covers the time range of 0.5-180 ms. Within 10 ms of the stepwise increase in Ca(2+) concentration, we identified a distinct compact conformational state with a drastically different molecular dimension. This process is too fast to study with a conventional stopped-flow apparatus. The compact conformational state was also observed without mastoparan, indicating that the calmodulin forms a compact globular conformation by itself upon Ca(2+) binding. This new conformational state of calmodulin seems to regulate Ca(2+) binding and conformational changes in the N-terminal domain. On the basis of this finding, an allosteric mechanism, which may have implications in intracellular signal transduction, is proposed.

[A case of mucinous tubular and spindle cell carcinoma of the kidney].

We report a case of mucinous tubular and spindle cell carcinoma (MTSCC) of the kidney. A 68-years old female consulted a primary hospital with a chief complaint of back pain. Computed tomography revealed the tumor of the left kidney, so she was referred to our department. The tumor, 45 x 42 mm in length, was slightly enhanced, and that had well-defined margins. We performed radical nephrectomy. Pathological findings suggested MTSCC. MTSCC is a rare type of renal cell carcinoma composed of a combination of low-grade tubular cuboidal cells and spindle cells in a mucinous stroma. The immunohistochemistry is effective in its diagnosis.

[Disseminated nocardiosis presenting as retroperitoneal abscess: a case report].

A 64-year-old man presented to our emergency room with right back pain on July 10, 2009. At the emergency room, abdominal enhanced computed tomography revealed a cystic lesion in the retroperitoneum. Then he was referred to our department. We performed percutaneous drainage of the retroperitoneal lesion and aspirated white pus. The retroperitoneal cystic lesion proved to be an abscess. Microscopic examination of a Gram stained specimen of the abscess revealed gram-positive bacillary fragments ; therefore, we suspected the pathogen to be Nocardia. He had a history of chronic glomerulonephritis and had received treatment consisting of 20 mg prednisolone, and 75 mg cyclosporine per day. He was regularly visiting the department of cardiovascular for follow-up of chronic heart failure. On the day before his visit to our emergency room, his chest X-ray medicine had revealed a nodular shadow. Then he was referred to the department of respiratory medicine and was scheduled to receive a bronchoscopy later. We suspected the nodule of the lung also to be an abscess of Nocardia. Later, head computed tomography (CT) revealed a brain abscess the pathogen of which was Nocardia. Nocardia is a filamentous, gram-positive, branched bacterium and classified as an aerobic actinobacteria. Nocardia species are difficult to diagnose due to non-specific clinical and histological manifestation. We report this case of disseminated nocardiosis presenting as retroperitoneal abscess. The disseminated nocardiosis was diagnosed without delay by percutaneous drainage and appropriate treatment was provided.

Non-native alpha-helix formation is not necessary for folding of lipocalin: comparison of burst-phase folding between tear lipocalin and beta-lactoglobulin.

Tear lipocalin and beta-lactoglobulin are members of the lipocalin superfamily. They have similar tertiary structures but unusually low overall sequence similarity. Non-native helical structures are formed during the early stage of beta-lactoglobulin folding. To address whether the non-native helix formation is found in the folding of other lipocalin superfamily proteins, the folding kinetics of a tear lipocalin variant were investigated by stopped-flow methods measuring the time-dependent changes in circular dichroism (CD) spectrum and small-angle X-ray scattering (SAXS). CD spectrum showed that extensive secondary structures are not formed during a burst-phase (within a measurement dead time). The SAXS data showed that the radius of gyration becomes much smaller than in the unfolded state during the burst-phase, indicating that the molecule is collapsed during an early stage of folding. Therefore, non-native helix formation is not general for folding of all lipocalin family members. The non-native helix content in the burst-phase folding appears to depend on helical propensities of the amino acid sequence.

Interactions responsible for secondary structure formation during folding of equine beta-lactoglobulin.

Equine beta-lactoglobulin forms a compact intermediate at an acidic pH (A state). It also forms an expanded and helical conformation at low temperatures (C state). The structure of a single disulfide mutant C66A/C160A is similar to the A state in the presence of salts, while it is similar to the C state at low anion concentrations. We have investigated the temperature-dependent change in the secondary structure using circular dichroism and proline scanning mutagenesis. At low anion concentrations, the helical content increased linearly as temperature decreased. In the presence of salts, the A state was cooperatively transformed into the C state at low temperatures. This suggests the importance of hydrophobic interactions for stabilizing the A state. Peptides encompassing native-like and non-native alpha-helices were synthesized to investigate the interactions responsible for helix formation in the A and C states. These did not form stable helices, indicating that not only the helices in the A state but also the helices in the C state are stabilized by long-range interactions. A longer fragment, CHIBL, which encompasses the structured region in the A and C states, showed a helical structure. Proline-substituted mutants of CHIBL showed CD spectral changes similar to the corresponding mutants of the full-length protein in the C state. Therefore, CHIBL has a structure similar to the corresponding region of the full-length protein in the C state. This result indicates that interactions responsible for helix formation in the C state reside in the sequence of CHIBL, and that the sequences outside CHIBL are essential for secondary structure formation in the A state.

Structural and thermodynamic consequences of removal of a conserved disulfide bond from equine beta-lactoglobulin.

A disulfide bond between cysteine 66 and cysteine 160 of equine beta-lactoglobulin was removed by substituting cysteine residues with alanine. This disulfide bond is conserved across the lipocalin family. The conformation and stability of the disulfide-deleted mutant protein was investigated by circular dichroism. The mutant protein assumes a native-like structure under physiological conditions and assumes a helix-rich molten globule structure at acid pH or at moderate concentrations of urea as the wild-type protein does. The urea-induced unfolding experiment shows that the stability of the native conformation was reduced but that of the molten globule intermediate is not significantly changed at pH 4 by removal of the disulfide bond. On the other hand, the molten globule at acid pH was destabilized by removal of the disulfide bond. This difference in the stabilizing effect of the disulfide bond was interpreted by the effect of the disulfide in keeping the molecule compact against the electrostatic repulsion at acid pH. In contrast to the wild-type protein, the circular dichroism spectrum in the molten globule state at acid pH depends on anion concentration, suggesting that the expansion of the molecule through electrostatic repulsion induces alpha-helices as observed in the cold denatured state of the wild-type protein.

[Synergetic responses after administration of interleukin-2 and Interferon-alpha combined with gamma knife radiosurgery in a patient with multiple lung and brain metastases: a case report].

A 51-year-old man with left renal tumor and multiple lung metastases was admitted to our hospital for treatment. Left nephrectomy was performed, and pathological diagnosis was renal cell carcinoma (clear cell carcinoma, G2, pT3a). Initially, Interferon-alpha (IFN-alpha) therapy was started for lung metastases. About 40 days after surgery, head magnetic resonance imaging revealed brain metastases, and therefore gamma knife radiosurgery(GKS) was performed. Since chest computed tomography showed no change in lung metastases, we tried a combination of interleukin-2 (IL-2) and IFN-alpha therapy to elininate those metastases. As a result, neither lung nor brain metastases could be detected at the 4th month follow-up examination. At 5 months after the IL-2 and IFN-alpha therapy, the patient attempted suicide. Therefore, the IL-2 and IFN-alpha therapy was stopped and an antidepressant was prescribed. Now 11 months after withdrawal of the IL-2 and IFN-alpha, the patient's mental condition remains stable. No recurrence of the cancer has been detected by CT.

Helical and expanded conformation of equine beta-lactoglobulin in the cold-denatured state.

The thermal unfolding transition of equine beta-lactoglobulin (ELG) was investigated by circular dichroism (CD) over a temperature range of -15 degrees C to 85 degrees C. In the presence of 2 M urea, a cooperative unfolding transition was observed both with increasing and decreasing temperature. The CD spectrum indicated that the heat and cold-denatured states of ELG have substantial secondary structures but lack persistent tertiary packing of the side-chains. In order to clarify the relation between the heat or cold-denatured state and the acid-denatured (A) state characterized previously, we have attempted to observe the temperature dependence of the CD spectrum at pH 1.5. The CD spectrum in the heat-denatured state is similar to that in the A state. The CD spectrum in the A state does not change cooperatively with increasing temperature. These results indicate that the heat-denatured state and the A state are the same structural state. On the other hand, the CD intensity at acid pH cooperatively increased with decreasing temperature. The CD spectrum at low temperature and acid pH is consistent with that in the cold-denatured state. Therefore, the cold-denatured state is distinguished from the heat-denatured state or the A state, and ELG assumes a larger amount of non-native alpha-helices in the cold-denatured state. Small angle X-ray scattering and analytical ultracentrifugation have indicated that ELG assumes an expanded chain-like conformation in the cold-denatured state in contrast to the compact globular conformation in the A state. The relation between the molecular size and the helical content in the partially folded states is discussed.

[Renal hemangiopericytoma discovered at a health screening: a case report].

We report a case of renal hemangiopericytoma which was incidentally discovered by ultrasonography at a health screening. A 58-year-old man was admitted to our hospital for close examination of the renal tumor. Computed tomography revealed the left renal tumor, 60 x 50 mm in size, which was well enhanced with contrast medium. Magnetic resonance imaging revealed an isointensity mass (T1-weighted) and high-intensity mass (T2-weighted) at the left kidney. Radical nephrectomy was performed on suspicion of left renal cell carcinoma. Histopathological examination revealed renal hemangiopericytoma. The present case is the 7th in the Japanese literature.

[Clinical study of male urethritis in Oogaki Municipal Hospital].

We studied 181 patients diagnosed with male urethritis at Oogaki Municipal Hospital from April 2002 to March 2004. Twenty-two out of 92 patients diagnosed with gonococcal urethritis (GU) and 52 out of 89 patients diagnosed with non-gonococcal urethritis (NGU) were positive for Chlamidia trichomatis by polymerase chain reaction (PCR). Most patients of male urethritis were in their twenties. Of GU patients, 39 (67%) were infected from commercial sex workers (CSWs). Of NGU patients, 12 (30%) were infected from CSWs, 24 (40%) from girl friends and 4 (10%) from their Twenty-eight (48%) out of GU patients were infected through oral sex. spouse. Eighty-three GU patients were treated with SPCM (2 g, one shot). Fifty-five patients could be evaluated for the efficacy of treatment. Elimination rate of Neisseria gonorrhoeae was 100% and 14 out of 18 patients with persisting urethritis had C. trichomatis. Eighty-two NGU patients were treated with minocycline, tosufloxacin, levofloxacin, gatiflixacin or clarithromycine. Sixty-six patients could be evaluated for the efficacy of treatment. Forty-one patients were diagnosed with non-gonococcal chlamydial urethritis (NGCU) and 25 patients were diagnosed with non-gonococcal, non-chlamydial urethritis (NGNCU). The clinical curative rate of NGCU and NGNCU was 93% (38/41) and 80% (20/25), respectively.

Thermal stabilization of penicillolysin, a thermolabile 19 kDa Zn2+-protease, obtained by site-directed mutagenesis.

Penicillolysin is a member of the clan MX and the family of M35 proteases. The enzyme is a thermolabile Zn(2+)- protease from Penicillium citrinum with a unique substrate profile. We expressed recombinant penicillolysin in Aspergillus oryzae and generated several site-directed mutants, R33E/E60R, A167E and T81P, with the intention of exploring thermal stabilization of this protein. We based our choice of mutations on the structures of homologous thermally stable enzymes, deuterolysin (EC 3.4.24.39) from A.oryzae and a peptidyl-Lys metallopeptidase (GfMEP) from the edible mushroom Grifora frondsa. The resulting mutant proteins exhibited comparable catalytic efficiency to the wild-type enzyme and some showed a higher tolerance to temperature.

Lack of association in Japanese patients between neuroleptic malignant syndrome and the TaqI A polymorphism of the dopamine D2 receptor gene.

OBJECTIVE: The molecular basis of neuroleptic malignant syndrome (NMS) is unclear, but clinical studies have noted a genetic predisposition. A recent genetic study suggested an association between NMS and the I A polymorphism in the dopamine D2 receptor (DRD2 ) gene. We further examined the association in a larger number of subjects. METHODS: We studied 49 Japanese patients previously diagnosed with NMS, and 123 schizophrenic patients treated with neuroleptics without occurrence of NMS. PCR and RFLP analyses were performed to screen the I A polymorphism. RESULTS: The I A1 allele frequency was 0.408 in NMS patients and 0.415 in patients without NMS. No significant differences in allelic or genotypic frequencies were observed between the two groups. CONCLUSIONS: We cannot conclude that the I A polymorphism is associated with development of NMS.