14-3-3ζ-Mediated Stimulation of Oxidative Phosphorylation Exacerbates Oxidative Damage Under Hypothermic Oxygenated Conditions in Human Renal Tubular Cells (HK-2).

Cellular survival and death are at least partially regulated by the phosphorylation of proteins. A chaperon protein, 14-3-3ζ, regulates the activity of many proteins by covering the phosphorylation site within a 14-3-3 binding motif. Therefore, regulation of 14-3-3ζ activity may affect the fate of cells subjected to cold preservation and/or hypothermic oxygenated conditions. The present study assessed whether 14-3-3ζ protects cells from hypothermic oxygenation-induced injury and clarified its role in mitochondrial functions. Human renal tubular cell line HK-2 or 14-3-3ζ-overexpressed HK-2 (ζHK-2) cells were subjected to 72 hours of normoxic cold preservation in UW solution with or without antioxidants and hydroperoxides. Cellular death, adenosine triphosphate (ATP) content, and MTT catabolism were evaluated. Deferoxamine treatment reduced cellular death and augmented ATP content in both cell types. These indices were higher in ζHK-2, regardless of deferoxamine treatment. Exposure to hydroperoxides did not affect cellular death in either cell type, whereas hydroperoxide supplementation significantly reduced ATP content, except for low-dose hydrogen peroxide in HK-2 cells. MTT assay at normal state showed higher values in ζHK-2 cells, whereas it was impaired by hydroperoxides in both cell types. These results suggest that accumulation of hydroperoxides as a byproduct of the augmented oxidative phosphorylation by 14-3-3ζ overexpression causes mitochondrial dysfunction. In conclusion, despite possessing many potentially protective functions, 14-3-3ζ exacerbates cellular injury under hypothermic oxygenated conditions. 14-3-3ζ accelerates mitochondrial functions together with iron-dependent oxidative damage. Although further investigations are necessary, upregulation of 14-3-3ζ could be a method to maintain mitochondrial function under hypothermic oxygenated conditions, as shown in hypothermic machine preservation of renal grafts, when appropriate antioxidant treatment is administered.

Long-term hepatic allograft acceptance based on CD40 blockade by ASKP1240 in nonhuman primates.

Blockade of the CD40-CD154 costimulatory signal is an attractive strategy for immunosuppression and tolerance induction in organ transplantation. Treatment with anti-CD154 monoclonal antibodies (mAbs) results in potent immunosuppression in nonhuman primates (NHPs). Despite plans for future clinical use, further development of these treatments was halted by complications. As an alternative approach, we have been focusing on the inhibition of the counter receptor, CD40 and have shown that a novel human anti-CD40 mAb, ASKP1240, markedly prolongs renal allograft survival in NHPs, although allografts eventually underwent chronic allograft nephropathy. On the basis of our previous findings that a CD40-CD154 costimulation blockade induces tolerance to hepatic, but not cardiac, allografts in rodents, we tested here our hypothesis that a blockade of CD40 by ASKP1240 allows acceptance of hepatic allografts in NHPs. A 2-week ASKP1240 induction treatment prolonged liver allograft survival in NHPs; however, the graft function deteriorated due to chronic rejection. In contrast, a 6-month ASKP1240 maintenance monotherapy efficiently suppressed both cellular and humoral alloimmune responses and prevented rejection on the hepatic allograft. No serious side effects, including thromboembolic complications, were noted in the ASKP1240-treated monkeys. We conclude that CD40 blockade by ASKP1240 would be a desirable immunosuppressant for clinical liver transplantation.

A notable case report of May-Hegglin anomaly with immune complex-related nephropathy: a genetic and histological analysis.

May-Hegglin anomaly (MHA) is a rare autosomal dominant disease characterized by macrothrombocytopenia and leukocyte inclusions with microfilaments in the ribosomes. Mutations in the MYH9 gene, encoding non-muscle myosin heavy chain IIA (NMMHC-IIA) have been identified in patients with MHA and other MYH9-related diseases. Two young males (an older and younger brother) presented with macrothrombocytopenia and leukocyte inclusion bodies. Electron microscopy (EM) revealed parallel filaments in leukocyte inclusion bodies characteristic of MHA. Immunofluorescence microscopy (IF) showed NMMHC-IIA antibodies in 1 - 2 leukocyte inclusion bodies. These findings were consistent with MHA and they were identified to express the MYH9 mutation, D1424H. The older brother underwent a renal biopsy because of persistent proteinuria. Histology revealed mesangial proliferative glomerulonephritis with granular deposits of IgG and C1q. EM showed that the dense deposits were located in subendothelial cells, mesangial cells and Bowman's capsule. Immunocytochemistry revealed that NMMHC-IIA antibodies were localized in podocyte and endothelial cells in the glomerulus. Moreover, the expression of nephrin and podocin, slit diagram protein, was normal. An inflammatory mechanism may occur separately from MYH9-related disease. This report presents a case of MHA with immune complex-related nephropathy.

[Relative size of the ventricle to the hemisphere in the neonatal MRI].

Using a software program for measuring surface area, we quantified the relative size of four parts of the lateral ventricles, including the body, the trigone, the anterior horn and the occipital horn, compared to the hemispheres in the axial plane of magnetic resonance imaging (MRI) in neonates. In 44 neonates without any neurological disorders from 26 to 41 weeks of gestational age (GA), MRI was performed between 12 and 124 postnatal days. The mean of the relative size of the ventricle compared to the hemisphere (RSVH) among the subjects showed a significant left-right asymmetry that was observed only in the occipital horn. However, in the body, the anterior horn and the occipital horn, the percentage of neonates with a larger left RSVH compared to the right RSVH was significantly higher than the percentage of neonates with a larger right RSVH compared to the left RSVH. The RSVH of the body and the occipital horn increased according to the number of postnatal days and decreased according to GA. Measuring RSVH was useful in assessing the size of the ventricle in the axial plane of neonatal MRI. It clarified the fact that normative asymmetry, GA of subject, and the number of postnatal days should be considered in assessing the size of ventricle.

[Factors contributing to exercise-induced hypoxemia in patients with chronic lung diseases].

Arterial oxygen tension (PaO2) was found to decrease linealy during incremental exercise in patients with chronic lung diseases. The slopes of PaO2 during increasing workload (delta PaO2/delta VO2) in patients with COPD were closely and negatively correlated with diffusing capacity of the lung (DLco). In patients with IPF, the values of delta PaO2/delta VO2 were much higher, and were more strongly correlated with DLco than those in patients with COPD. Therefore, exercise-induced hypoxemia (EIH) was found to be related to three factors: PaO2 at rest, PaO2 slope predicted from %DLco, and exercise workload. The most important factor in EIH in patients with COPD was the decrease in PvO2 during exercise, which was caused by right heart failure and venous admixture. In patients with IPF, EIH was caused by abnormal O2 diffusion during exercise, decreased PvO2, and venous admixture. In conclusion, the factors contributing to EIH depend on the workload, so exercise testing is needed to decide on exercise therapy and O2 therapy in patients with chronic lung diseases.

Relationship between exercise capacity and the severity of emphysema as determined by high resolution CT.

We investigated the relationship between exercise capacity or exercise-induced hypoxaemia and the severity of pulmonary emphysema in 20 patients with pulmonary emphysema. The patients underwent pulmonary function tests, high resolution computed tomography and incremental treadmill exercise testing. Computed tomography scans were obtained at four levels in the lungs, and emphysema scores were determined by the visual assessment of low attenuation areas as a measure of the severity of parenchymal destruction. The emphysema score correlated significantly with diffusing capacity (r = -0.69) in the pulmonary function tests. Among the exercise test parameters, the emphysema score correlated significantly with the total distance walked (r = -0.74), with maximal oxygen consumption (VO2max) (r = -0.77), with arterial oxygen tension (PaO2) at rest (r = -0.50) and at the maximum workload (r = -0.58), and with the decrement of arterial oxygen tension per litre of oxygen consumed (r = -0.64). These results suggest that exercise capacity and exercise-induced hypoxaemia are related to the extent of destruction of lung parenchyma in patients with pulmonary emphysema.

Development of ultradian rhythm of EEG activities in premature babies.

Ultradian rhythms in electroencephalogram (EEG) activity in premature neonates were studied. The values of square roots of total power (SQP) of EEG showed a normal Gaussian distribution. We analyzed the smoothed time series of SQP by autoregressive analysis and component analysis. Ultradian rhythm was divided into four groups by period length (group I: 5-30 min, group II: 30-60 min, group III: 60-100 min, group IV: longer than 100 min). Group IV rhythms were not detected before 32 weeks of gestation. Changes in the characteristic values of ultradian components in group II showed that there were critical periods in the development of central nervous activity at 29 and 33 weeks of gestation. This method allowed detection of ultradian rhythms, even at very early gestation, directly from the neonatal central nervous system. This method may be applicable to ill infants.

[Quantification of electroencephalogram continuity by 24-hour recording in very premature infants].

We aimed to quantify electroencephalogram (EEG) continuity for 24 hours in very premature infants. A total of 122 days of continuous two-channel EEG recordings were performed in 28 premature infants from 26 to 33 weeks of conceptional age (CA). None of the infants showed any evidence of neurological impairments during the course of their hospitalizations and showed normal neurological outcome. The 24-hour EEG recording was divided into 5.5-minute periods. The EEG of each period was classified into five EEG categories according to EEG continuity, and the percentage of each of them during the 24-hour recording was calculated. The percentages of continuous and continuous dominant EEG categories were increased with increasing CA. On the other hand, the percentages of discontinuous and discontinuous dominant EEG categories were decreased with increasing CA.

[Evaluation of physical fitness and exercise performance in patients with chronic pulmonary emphysema].

Physical fitness was studied in patients with chronic pulmonary emphysema using Kraus-Weber methods in addition to pulmonary function and exercise tolerance. In Kraus-Weber tests, explosive strength of abdominal muscles in these patients were within the normal range, but both abdominal and back muscle endurance were significantly diminished compared to age-matched controls. On the other hand, flexibility was not different between the patients and the controls, although large variation was present. Exercise performance as assessed by 6 minutes' walk distance in patients was significantly correlated with FEV1.0, DLco and maximal inspiratory mouth pressure, as well as explosive strength of abdominal muscles and abdominal and back muscle endurance capacity. Treadmill walking training for 20 minutes with a load greater than 80% VO2max, twice a week for 2 months was performed in 11 patients with mild to moderate pulmonary emphysema. Six minutes' walk distance (6MD) was significantly prolonged with improvement of back muscle endurance and flexibility. Another walking training consisting of five repetitions of two minutes' near maximal walking and a two minute interval of rest was performed in 6 patients with severe pulmonary emphysema. 6MD tended to increase with improvement of both back and abdominal muscle endurance. However, pulmonary function tests and VO2max showed no significant changes after both types of training. Improved walked distance after the training was significantly correlated with improved VO2 at AT. Furthermore VO2, VE, HR and lactate production during exercise at the same load were significantly decreased compared to pre-training. Dyspnea sensation measured by modified Borg scale during exercise was improved after the training. It is concluded that a physical training program adapted to the condition of the individual patients could improve exercise performance, and should be prescribed in addition to medication.

[Dyspnea and ventilatory muscle function during exercise on air and oxygen breathing in patients with chronic obstructive pulmonary disease (COPD)].

Exercise performance and dyspnea in COPD patients have been shown to be improved with supplemental oxygen, although the exact mechanisms resulting in the improvement are still unclear. The purpose of the present study was to investigate a possible relationship between ventilatory muscle function and sensation of dyspnea (modified Borg Scale) during exercise on 20% O2 and 30% O2. Eight patients with COPD (FEV1 1.06 L +/- 0.30 L) exercised on a treadmill two times breathing compressed air or 30% oxygen with a one hour rest in between. The electrical activity of the diaphragm (EMGdi) was recorded with esophageal electrodes, and that of the sternomastoid muscle (EMGsm) was recorded from the fine wire electrodes. The ratio of high frequency (150 to 350 Hz) to low frequency (20 to 47 Hz) power (H/L) of EMGdi was analyzed to assess diaphragmatic fatigue, which was defined as a 20% fall of H/L ratio from the control value. Flow, volume, O2 Saturation (SaO2), esophageal pressure (Pes) and transdiaphragmatic pressure (Pdi) were measured. Tension time index (TTdi) was calculated from Pdi and the ratio of inspiratory time to total time for one cycle (Ti/Ttot). At rest, we measured maximal esophageal pressure (Pesmax), maximal transdiaphragmatic pressure (Pdimax), maximal integrated EMGdi (EMGdimax) and EMGsm (EMGsmmax). Incremental exercise was discontinued by dyspnea. The walking distance achieved was increased in all patients on 30% O2. Dyspnea and desaturation were significantly improved on 30% O2 breathing, and the onset of diaphragmatic fatigue was delayed.(ABSTRACT TRUNCATED AT 250 WORDS)

[Relationship between anaerobic threshold and breathlessness during exercise].

We studied the possibility that during incremental exercise anaerobic threshold (AT) is associated with an increase in breathlessness in subjects with increased impedance or respiratory system. Six patients with chronic lung diseases (CLD) and six healthy subjects performed cycle ergometer exercise under two conditions; with free resistive loading and with 9 cmH2O/l/sec of inspiratory resistive loading (IRL). Ventilation, respiratory pattern, VO2, VCO2, were measured every 15 sec by a metabolic cart, Minato Respiromonitor RM 300. AT was determined by V-slope method. Mouth occlusion pressure (P0.1) was also measured in six subjects during exercise. The subjects selected a number of modified Borg scale (psi) to express their breathlessness during exercise by pushing a electrically potentiated remote button. Slopes of changes in psi with increasing work-load, delta psi/delta watt-slope, were calculated before and after V-slope AT, and each inflection point of VE, respiratory rate, VT/Ti, and VCO2. In order to evaluate which parameters are the most responsible for a change in psi, statistical significance (P-value) in changes of slope of each parameters were compared. The delta psi-slope increased with the highest significance after AT point. Under IRL the significance was more pronounced. There was a highly significant linear correlation between P0.1 and psi during exercise both under free resistive loading and IRL. In addition the increase of P0.1 with incremental exercise was characterized by a presence of inflection at a workload of V-slope AT in most of the subjects. From these results it was concluded that anaerobic threshold was closely associated with an increase of breathlessness during exercise via enhancing respiratory motor command.(ABSTRACT TRUNCATED AT 250 WORDS)

Sequential changes in electroencephalogram continuity in very premature infants.

This study was conducted to quantify sequential changes in electroencephalogram (EEG) continuity for 24 h in very premature infants. For a total of 122 days, continuous 2-channel EEG recording was conducted for 28 premature infants from 26 to 33 weeks of conceptional age (CA). None of the infants showed evidence of neurological impairment during hospitalization. Normal neurological outcome was noted at a minimum 12 months of age. By classifying each 5.5 min epoch according to EEG continuity, the number of contiguous epochs of each series of discontinuous type (DTs) and the number of epochs between two series of discontinuous type (IDTIs) were counted at each CA. The duration of DT decreased with increasing CA. The mean duration remained at 13-16 min after 29 weeks CA. The mean duration of each IDTI increased with CA, up to about 1 h at 33 weeks. A constant period of DTs was noted at longer intervals with increasing CA. These changes appear related to the development of sleep state organization with CA.

[Urinary cytology studied during 10 days after transurethral resection of bladder tumour and its relation to tumour recurrence].

Whether or not recurrence is related to the results of urinary cytology examined within 10 days after transurethral resection of bladder tumour was studied retrospectively in 47 patients with superficial bladder tumour. Of 7 cases with positive cytology during the postoperative 10 day period, 4 cases had a later recurrence of tumour and 2 cases had a residual tumour due to incomplete resection of original tumour. By microscopic chromocystoscopy, in 11 patients concurrent urothelial atypia (carcinoma in situ or dysplasia) was found in the apparently normal mucosa. Nine of the 11 cases had a later recurrence of tumour or a residual tumour. Of in total 15 patients combined with abnormal cytology and concurrent urothelial atypia, 12 (80%) were found with recurrence of tumor cystoscopically 4 approximately 64 months (mean; 20.6 months) after TUR. This recurrence rate was significantly (p less than 0.05) higher than that (42.4%) in patients without positive cytology and concurrent urothelial atypia. These results suggest that positive urinary cytology during 10 days subsequent to TUR and/or association with concurrent urothelial atypia are indicative of later tumour recurrence, incomplete resection of tumour or presence of additional occult tumour foci.

[Hairy cell leukemia expressing SIgM+, SIgG-, CD11b+ and CD21+ and accompanying lymphadenopathy without splenomegaly].

Hairy cell leukemia (HCL) expressing both surface monocytoid antigen and IgM (kappa) was reported. A 62-year-old male was admitted to our hospital in September 21, 1989 because of leukocytosis. Physical examinations showed axillary and inguinal lymphadenopathy but no hepato-splenomegaly. The leukocyte count was 12,600/microliters with 73% of abnormal cells like large lymphocytes which had abundant cytoplasm and hairy appearance under phase microscopy. They had ruffles with microvilli under electron microscope. Bone marrow puncture showed normocellular marrow with 71.2% of abnormal cells similar to the peripheral blood. Surface markers were CD11b+, CD21+, HLA-DR+, Tac- and IgM (kappa). They were positive for ++acid phosphatase staining, but negative for peroxidase and tartrate-resistant acid phosphatase staining. He was diagnosed as Japanese type HCL. HCL expressing both surface monocytoid antigen and IgM is rare and the clinical features of our case are compared with those reported in Japan.

Micro-enzyme immunoassay of vasoactive intestinal polypeptide (VIP)-like immunoreactive substance in bovine milk.

A sensitive and specific enzyme immunoassay for vasoactive intestinal polypeptide (VIP)-like immunoreactivity was developed with the use of synthetic carboxy-terminal (C-terminal) fragment (residue 11-28) of porcine VIP conjugated with beta-D-galactosidase and a second antibody-coated immunoplate. Using 4-methylumbelliferyl beta-D-galactopyranoside as a fluorogenic substrate, the minimum amount of VIP-like immunoreactive substance (VIP-IS) detectable by this method was 0.1 fmol/well (2.5 pmol/l). The level of VIP-IS in bovine foremilk was above 100 pmol/l, which was more than eightfold higher than that in normal milk.

Increased transport of theophylline into gastrointestinal lumen and gastrointestinal dialysis by activated charcoal in rats with hepatic cirrhosis.

The characteristics of exsorption and/or excretion of theophylline into the small intestinal lumen in rats with hepatic cirrhosis (HC rats) induced by carbon tetrachloride were investigated by an in situ single-pass perfusion technique. The serum concentrations of theophylline after i.v. administration of aminophylline (10 mg/kg) in the HC rats were significantly higher than those in normal rats during the experimental period. Moreover, the exsorption of theophylline from blood into the intestinal lumen was significantly increased in the HC rats compared with the normal rats. Treatments with oral activated charcoal reduced the serum theophylline levels in the HC rats. Consequently, gastrointestinal dialysis by oral administration of activated charcoal may be a useful method to remove poisonous drugs from the blood in patients with hepatic failure (including cirrhosis), which decreases the systemic clearance.

[Developmental characteristics of the alpha waves of EEG in normal healthy children].

The records of waking state electroencephalography (EEG) of 1,340 normal children aged from 3 to 12 years were subjected to analysis using an autoregressive model. The results were summarized as follows. (1) 6,814 components of second-order activities were obtained through component analysis of the 1,218 EEG records. The frequency polygons of damping frequency of the second-order component waves verified several modes, each of which was enhanced in the eight frequency ranges. (2) The average percent-power of the alpha-1 waves decreased with increasing ages from 3 years, whereas that of the alpha-2 waves increased. That of the alpha-3 waves showed no change with ages. (3) The average damping times of alpha-2 waves increased with increasing ages. (4) Dominancy in frequency showed a course of two step increments at 3-6 and 10 years of age. The former was chiefly due to the increase in the alpha-1 wave and the latter in the alpha-2 wave. There were no significant increases of alpha-3 waves. (5) By means of EEG pattern discrimination, the differences in alpha components among different age groups were obtained as generalized (Maharanobis') distances. The alpha-1 activity showed a significant difference in amount between the age group of 3-6 years and the other age groups. The alpha-2 activity showed a significant difference in amount between the age group of 10 years and the other age groups. (6) The development of brain activity proceeds faster in the posterior areas and relatively slower in the central areas.

[Developmental characteristics of the beta waves of EEG in normal healthy children].

By the use of an autoregressive (AR) model, development of beta-waves in quantitative EEG parameters was studied in 1,340 normal children aged from 3 to 12 years. The results were summarized as follows. The beta band(13.5-25 Hz) was divided into three frequency ranges; beta-1(13.5-17.4 Hz), beta-2(17.5-21.4 Hz) and beta-3 (21.5-25 Hz) waves. The average power and percent-power of the beta-1 and beta-2 waves increased with advancing age, whereas these of the beta-3 wave showed no change with advancing age. By means of EEG pattern discrimination, critical period of beta band in the developing EEG was observed at the age of 4 years in frontal region, 10 years in central region and 3 to 7 years in occipital region. Those results suggest that the development of beta activity may be faster in frontal, occipital areas and relatively slower in central area.

[An application of autoregressive model to pattern discrimination of brain electrical activity mapping].

We developed a method of illustrating significant regional differences between two records of brain electrical activity mapping (BEAM), using pattern discrimination of autoregressive (AR) EEG analysis. In this study, the EEG data could be objectively reduced to a few AR coefficients, and this statistical method was successfully applied to discrimination of differences in BEAM among records from two or more subjects. A significant difference was illustrated at the right parietal region between two age groups, 6 years and 8 years, in normal children. It was concluded that this new approach of BEAM analysis through a statistical taxonomy was clinically useful for evaluation of the normal development of BEAM in children and for localization of functional brain abnormalities.