A novel predictive method for assessing the quality of isolated pancreatic islets using scanning electrochemical microscopy.

INTRODUCTION: The current methods for evaluating islet potency are not useful in clinical transplantation. Therefore, we need reliable, rapid methods enabling accurate prediction of islet quality. MATERIALS AND METHODS: We evaluated respiratory activity using scanning electrochemical microscopy (SECM), glucose-stimulated respiratory activity, glucose-stimulated insulin release, ADP/ATP assays, insulin/DNA levels, and Trypan blue exclusion tests as predictive methods for the ability of isolated rat islets to cure syngeneic diabetic rats. RESULTS: Although glucose-stimulated respiratory activity, basal respiratory activity, ADP/ATP ratio, and glucose-stimulated insulin release were significantly correlated with the outcome of transplantation into diabetic rats, there was no correlation between outcomes, insulin/DNA ratios, and Trypan blue exclusion tests. The glucose-stimulated respiratory activity in islet preparations that could cure diabetic rats was significantly greater than those unable to cure diabetes. Rat islets with >1.5-fold glucose-stimulated respiratory activity consistently cured diabetic rats, whereas those with a value <1.5 hardly cured any rats. CONCLUSION: Measurement of the glucose-stimulated respiratory activity using SECM technique is a novel method that may be useful as a rapid, potent predictor of the outcome of clinical islet transplantation.

Permeation of redox species through a cell membrane of a single, living algal protoplast studied by microamperometry.

Permeation of several redox species through a cell membrane of a single algal protoplast (radius 100 microns) was investigated by amperometry with a Pt microdisk electrode (disk radius, 6.5 microns) located near the membrane. The redox current observed at the microelectrode decreased as the microelectrode approached the cell membrane since the membrane acted as a barrier for diffusion of redox species from bulk to the microelectrode. Permeability coefficient (Pm) of the protoplast membrane was determined by the quantitative analysis of the variation of the redox current with microelectrode-membrane distance using digital simulation. The Pm values for Fe(CN)6(4-), Fe(CN)6(3-), Co(phen)3(2+), ferrocenyl methanol(FMA) and p-hydroquinone(QH2) were < or = 1.0 x 10(-4), < or = 1.0 x 10(-4), 1.0 x 10(-3), 5.0 x 10(-3) and 2.0 x 10(-2) cm/s, respectively. Using these Pm values, the concentration changes inside a model cell and chloroplast were theoretically calculated.

Microring-ring electrode for manipulation of a single cell.

Dielectrophoretic manipulation of a single myeloma cell was carried out using microring-ring electrodes (tip radius, 3-5 microns). When a.c. voltages were applied, the cell nearby the electrode tip was forced to move and trapped at the tip due to dielectrophoretic force induced by locally intense electric field.

Electron transferase activity of diaphorase (NADH: acceptor oxidoreductase) from Bacillus stearothermophilus.

Electrochemical kinetic measurements were carried out for electron-transfer between NADH and the oxidized forms of mediators (ferrocenylmethanol (FMA), ferrocenyl-1-ethanol (FEA), N,N,N',N'-tetramethylphenylenediamine (TMPD), Co(Phen)2+(3) and Fe(CN)4-(6)) catalyzed by diaphorase (NADH: acceptor oxidoreductase, EC 1.6.99.-) purified from Bacillus stearothermophilus. Cyclic voltammograms for the mediators with excess NADH in the presence of diaphorase gave steady-state currents. The quantitative analysis of the dependence of the current on the mediator concentration yielded a Michaelis constant (Km) and molecular activity (ko), which are difficult to determine by the conventional spectrophotometric method. Small Km and large ko values were observed for the oxidized forms of FMA, FEA and TMPD compared to those for Co(Phen)3+(3) and Fe(CN)3-(6). It is suggested that the reaction pocket of the present diaphorase is hydrophobic. The present electrochemical procedure for the determination of the kinetic parameters is applicable widely to similar enzyme reactions.

In vivo analysis of hydrogen peroxide and lipid radicals in the striatum of rats under long-term administration of a neuroleptic.

It has been hypothesized that free radicals play a causative role in tardive dyskinesia, which is an inveterate movement disorder caused by chronic administration of neuroleptics. To verify this hypothesis, rats were reared while being regularly treated with water containing a neuroleptic, haloperidol (HPD), for 1 year (HPD group). The changes in the striatal hydrogen peroxide content of the rats in the HPD and control groups were measured by using a Pt-disk microelectrode while the animals were in a freely moving state following intraperitoneal administration of HPD (HPD challenge). We also performed electron spin resonance (ESR) detection of lipid radicals in the striatum before the HPD challenge. HPD challenge led to significant elevation of the intrastriatal hydrogen peroxide in all animals, but the elevation in the HPD group was smaller than that in the control group. However, in the HPD group, marked ESR signals of intrastriatal lipid radicals were observed. We think that these results support the hypothesis on the role of free radicals in tardive dyskinesia.

Hydrogen peroxide augmentation in a rat striatum after methamphetamine injection as monitored in vivo by a Pt-disk microelectrode.

We fabricated a Pt-disk microelectrode (diameter 30 microns) to conduct differential double-pulse amperometry (first step: 750 mV, 1 s; second step: 1,100 mV, 1 s) to detect hydrogen peroxide in the brain of a freely moving animal. This measurement determined hydrogen peroxide (detection limit, 0.03 microM) without any observable influence from other oxidizable species, such as dopamine (DA), ascorbic acid, or uric acid. The electrode was implanted into the right striatum of a rat. After intraperitoneal injection of methamphetamine (MAP), hydrogen peroxide concentrations were directly detected while the behavioral changes were monitored. MAP injection led to significant augmentation of hydrogen peroxide, the elevation of which depended on the dose of MAP. This is consistent with a previous report on the increase of DA-release caused by amphetamines and indirect evidence of the production of hydrogen peroxide via DA-metabolism.

Reduced-dose chop therapy for elderly patients with non-Hodgkin's lymphoma.

While CHOP therapy is effective for malignant lymphoma, the optimum schedule for elderly patients remains controversial. The present study investigated the usefulness of reduced-dose CHOP therapy for elderly patients. Previously untreated patients aged 65 years or older with intermediate to high-grade non-Hodgkin's lymphoma were given up to 6 courses of reduced-dose CHOP therapy at 3-week intervals. Group A patients were given (5/6) of the standard dose and Group B received 7/12 of the standard dose. Filgrastim was administered when the white blood cell count fell below 2,000/microL. Fifty-seven patients were evaluable and the scheduled therapy was completed in 37. For patients aged from 65 to 79 years and for patients older than 80 years, the complete response rate was 79.5% and 46.2%, overall 3-year survival was 58.2% and 30.4%, and event-free 3-year survival was 49.3% and 44.4%, respectively. Major toxicities (> or = grade 3) included leukopenia in 42 patients and documented infection in 7 patients. Grade 3 cardiac plus renal failure, grade 3 peritonitis due to small bowel perforation, and grade 3 liver dysfunction occurred in 1 patient each. One patient died of toxicity (grade 4 hematological toxicity and pneumonia). In conclusion, it seems that in the elderly patients with non-Hodgkin's lymphoma, response to reduced-dose ((5/6) dose) CHOP therapy is comparable to that for standard CHOP in younger adults, mainly because of improved dose-intensity.

Real-time multisite observation of glutamate release in rat hippocampal slices.

A multichannel glutamate sensor was fabricated that consists of enzyme modified electrodes and has a high sensitivity and selectivity to glutamate. We placed a rat hippocampal slice on the sensor and monitored the current at four electrodes resulting from the stimulation with muscimol, a gamma-aminobutyric acid(A) (GABA(A)) receptor agonist. We obtained different glutamate concentration increases at the different positions, suppressed by bicuculline, a GABA(A) receptor antagonist. This demonstrated that the sensor can monitor the glutamate released via GABA(A) receptors pathways, and the difference in the concentrations may indicate differences in the distribution of GABA(A) receptor as well as diverse receptor functions. This multichannel sensor may be useful for non-invasive, real-time monitoring of glutamate distribution, which would make it a valuable tool for pharmacological analysis.

Dielectrophoretic manipulation of a single chlorella cell with dual-microdisk electrode.

Dielectrophoretic manipulation of a single chlorella cell was performed using a dual-microdisk electrode, which consists of two Pt-Rh ultrafine wires (ca. 1-microm radius) sealed in a glass capillary. An attractive or repulsive force was induced on the chlorella depending on the frequency of the ac voltage applied between the two disk electrodes. To avoid the direct contact of a chlorella with the metal, a dual electrode with retracted disks was fabricated and used for forming a micropattern of chlorellas at a solid substrate. The effect of both the frequency and ion concentration of the solutions on the dielectrophoretic force exerted on a chlorella cell was investigated in detail based on the theories of dielectrophoresis.

Mapping peroxidase in plant tissues by scanning electrochemical microscopy.

Scanning electrochemical microscopy has been firstly used to map the enzymatic activity in natural plant tissues. The peroxidase (POD) was maintained in its original state in the celery (Apium graveolens L.) tissues and electrochemically visualized under its native environment. Ferrocenemethanol (FMA) was selected as a mediator to probe the POD in celery tissues based on the fact that POD catalyzed the oxidation of FMA by H(2)O(2) to increase FMA(+) concentration. Two-dimensional reduction current profiles for FMA(+) produced images indicating the distribution and activity of the POD at the surface of the celery tissues. These images showed that the POD was widely distributed in the celery tissues, and larger amounts were found in some special regions such as the center of celery stem and around some vascular bundles.

Infectivity and oocyst excretion patterns of Cryptosporidium muris in slightly infected mice.

To determine the infectivity of Cryptosporidium to hosts in slight infections, we examined the infectivity and oocyst output patterns of Cryptosporidium muris in mice inoculated with small numbers of oocysts. One of the 25 ICR mice inoculated with 2.4 x 10(1) oocysts and 19 of the 25 mice inoculated with 2.4 x 10(2) oocysts shed oocysts in the feces after inoculation. Four of the 50 mice inoculated with 2.4 x 10(1) oocysts for 10 consecutive days also shed oocysts and their OPG values were similar to that of the mice which received 2.4 x 10(2) oocysts. Consequently, it is clear that less than 10% of the mice which received 2.4 x 10(1) C. muris oocysts for 10 consecutive days.

[Comparison between two systems for assaying the level of antibody against Streptococcus agalactiae (GBS) type III].

Levels of antibody against GBS type III in pregnant women have been determined using ELISA assay method that we originally deviced. Toyo Jozo Co. have also introduced an assay system (the Toyo kit) that resembles ours. The present study was performed to compare our system with the Toyo kit. Correctivity between the two systems was good, giving a regression curve of Y = 1.18X - 0.60 and a correlation coefficient of r = 0.794. Antibody levels in pregnant women were determined by the Toyo kit, and were found to be less than 10 micrograms/ml in almost all samples. Among 19 serum samples from vaginal carriers of GBS type III, the antibody levels ranged from 1.5 to 93.8 micrograms/ml. Among seven maternal serum samples in cases of GBS type III infection, the maximum level was 6.0 micrograms/ml. Thus, the Toyo kit showed satisfactory sensitivity for determining the level of antibody against GBS type III. Hereafter, we intend to use the Toyo kit for determining antibody levels in pregnant women.

[Usefulness of the mailed specimens for mass examination of Chlamydia trachomatis and Neisseria gonorrhoeae with EIA].

The infections with C. trachomatis and N. gonorrhoeae have recently been determined routinely with the commercial kits for detecting the antigens of these organisms in both the clinical and mass examination laboratories. In mass examination, a change during transporting the specimens must be avoided. Hence, a study on whether the antigens were changed with atmospheric temperature was carried out. As for C. trachomatis, no change in antigen was found on the enzyme immunoassay (EIA) absorbance of the antigen quantity corresponding to the cell of 4.8 x 10(4), 2.4 x 10(4) and 6.0 x 10(3) (IFU/ml) until 5 days at 4, 25 and 37 degrees C respectively after sampling. The coefficient variation was found out to be ca. 10%. In a test on antigen stability of N. gonorrhoeae, similar results to that of C. trachomatis were obtained on the following antigen quantity: 2.4 x 10(3), 3.3 x 10(2) and 9.0 x 10 (CFU/ml) under the same conditions as in the above. The coefficient variation was found out to be 10% or less. To investigate whether there is any difference on the stability of C. trachomatis antigen between mailing and hand carrying, a total of 133 specimens collected from many clinics were subjected to the detection of antigen with the commercial EIA kits. The results of comparison on both transport methods were as follows: agreement rate 96.2%, positive rate 100% and negative rate 95.2%. The highly correlation between the both transportation was confirmed in the detection of antigen; that is, Y = 1.03X + 0.03, r = 0.936.(ABSTRACT TRUNCATED AT 250 WORDS)

[Epidemiological survey for hemolytic streptococci isolated from children in Tokyo].

During the 20-year period between 1979-1998, a total of 4,176 strains of hemolytic streptococci have been isolated from 20,118 healthy primary school children and little children in Tokyo Metropolitan (Tokubetsuku, Tama and Tosho). Culture of throat swabs every November and the following February during the 20-year period were made and serological grouping and typing for isolates were done by T agglutination method. The results were as follows. 1) Serological group of hemolytic streptococci isolated from children were 3,188 strains (76.3%) for isolates of group A out of total strains of 4,176, 569 strains (13.6%) for isolates group B, 63 strains (1.5%) for isolates of group C and 356 strains (8.5%) for isolates of group G. 2) The most dominant was T12 during 1979-1998, and other relatively frequent serotypes were T28, T1, T4, T6 in that order. These ranks of and the main epidemic serotypes showed a similar trend in the 3 areas. 3) The isolation rates of group A streptococci were 15.9% in Tokubetsuku, 17.1% in Tama and 14.9% in Tosho. The average of 3 areas were 15.8%. 4) The epidemic cases seemed to be caused by group A streptococci were 20 cases, their isolated serotype were 7 cases by T28, 5 cases by T12, 4 cases by T6, 2 cases by T4, each 1 case by T1 and T25. 5) A total of 2,927 strains of group A streptococci were examined for drug sensitivity. All strains were sensitive to beta-lactam group of antibiotics (benzylpenicillin and cephaloridine). Resistant (MIC > or = 25 micrograms/ml) to TC, CP and EM etc. were 740 strains (25.3%) in this study. The incidence of resistant strains were to TC 493 strains (66.6%) out of 740 strains, 81 strains (10.9%) for TC.CP, 72 strains (9.7%) for EM and 66 strains (8.9%) for TC.CP.EM.OL.LCM. TC resistant strains have not varied much through the whole period, but CP and EM resistant strains were very variable by year. Many resistant strains to TC were T4, to EM and multiple drug resistant were T12. 6) The rates of isolates of the same type of group A streptococci in school child individual during for the tests taken twice a year were 12.3%, indicating group A streptococci, according to the duration of the carrier state, seems to be a short period.

[Epidemiological study on Chlamydia trachomatis infection in obstetrics and gynecology field in Tokyo].

In conjunction with the Tokyo Branch of Japan Association for Maternal Welfare, we have been carrying out examinations of Chlamydial infection on women, especially pregnant women, living in Tokyo. Specimens were collected from 212 gyneco-obstetric clinics in Tokyo during 4 years from January 1987-December 1990. A total of 13,925 swab specimens from patients who were suspected of sexually transmitted diseases from clinical findings and pregnant women who requested the examination were tested for the presence of antigen to C. trachomatis with EIA (Chlamydiazyme). Epidemiological analysis based on the results and case cards that were described sex, age, occupation and clinical findings were conducted. The results obtained are briefly summarized as follows. 1) C. trachomatis antigen was detected in 12.8% (1,237/9,657) of the female patients, 31.0% (124/400) of the male patients and 6.8% (10/168) of the infants. The detection rates of C. trachomatis antigen in the male patients was significantly higher than that in the female and the infants patients. C. trachomatis antigen was detected in 6.1% (226/3,683) of pregnant women. 2) The detection rates of C. trachomatis antigen were compared by occupation of female patients. Most high rate was 26.6% (53/199) in bar hostess , subsequently 20.1% (102/508) in students, 19.8% (59/298) in prostitutes, 13.6% (455/3,348) in office girls and 7.2% (471/6,573) in house-wives. It was noteworthy that the detection rates of C. trachomatis antigen in students was as similarly high as that in prostitutes. 3) The detection rates of C. trachomatis antigen was also compared by age groups. The detection rate had a tendency to be high in younger people.(ABSTRACT TRUNCATED AT 250 WORDS)

[Molecular analysis of bcr/abl mRNA in chronic myelogenous leukemia after bone marrow transplantation by using RT-PCR method].

Thirty three patients with chronic myelogenous leukemia (CML) treated by allogeneic bone marrow transplantation (BMT) were evaluated for bcr/abl mRNA using the reverse transcriptase-polymerase chain reaction (RT-PCR). The bcr/abl mRNA was detected in 22 out of 33 patients in clinical complete remission after BMT. The bcr/abl mRNA was present only transiently in 6 patients. It was speculated that leukemia cells were not eradicated by conditioning therapy of BMT, but patients maintained clinical complete remission due to GVL (graft versus leukemia) effect. Further study is necessary to estimate the clinical value of this technique to predict the outcome in CML patients.

Spontaneous remission of epstein-barr virus-positive diffuse large B-cell lymphoma of the elderly.

A 94-year-old female patient presented with anorexia and left axillar lymphadenopathy on admission. Her past history was angina pectoris at 83 years of age and total gastrectomy due to gastric cancer at 87 years. The family history revealed that her son had had a malignant lymphoma, the histopathological diagnosis of which was diffuse large B-cell lymphoma. A physical examination showed both cervical, axillar, and inguinal lymphadenopathy without tenderness. She had elevated lactate dehydrogenase, ferritin, and soluble interleukin-2 receptor (sIL-2R). Whole-body computed tomography confirmed the cervical, axillary, and inguinal lymphadenopathy. Gallium-68 imaging revealed positive accumulation in these superficial lymph nodes. A right inguinal lymph node biopsy showed features of Epstein-Barr virus-associated lymphoproliferative disorder. Immunohistological studies on this lymph node biopsy showed CD20-positive large cells, CD3-positive small cells, and CD30-partly-positive large cells. In situ hybridization showed Epstein-Barr virus-positive, LMP-partly-positive, and EBNA2-negative cells. She refused chemotherapy as her son had died from hematemesis during chemotherapy. She received intravenous hyperalimentation for 1 month after admission. No palpable lymph nodes were identified by physical examination or computed tomography 3 months after admission, and regression of lactate dehydrogenase, ferritin, and sIL-2R was observed. She recovered from anorexia and was discharged. She died from pneumonia 10 months later after initial symptoms of anorexia. The autopsy showed no superficial lymphadenopathy.

Detection of microspotted carcinoembryonic antigen on a glass substrate by scanning electrochemical microscopy.

Microspots of carbinoembryonic antigen (CEA) on glass substrates were characterized by scanning electrochemical microscopy (SECM). CEA was immobilized via a sandiwch method using horseradish peroxidase (HRP)-labeled anti-CEA. The reduction current of the oxidized form of ferrocenylmethanol generated by the HRP reaction was monitored to view SECM images. This method detects as low as ∼10(4) CEA molecules in a single 20-µm-radius spot.

A microvoltammetric study of permeation of ferrocene derivatives through a planer bilayer lipid membrane.

Permeability of ferrocene derivatives through a planer bilayer lipid membrane (BLM) was examined by an electrochemical method using microelectrodes. Location of the microelectrode tip inside the unstirred layer enables the detection of electroactive substances permeating the membrane without unstirred layer perturbation.

Microamperometric measurements of photosynthetic activity in a single algal protoplast.

The effects of p-benzoquinone (BQ) on photosynthetic and respiratory electron transport in a single algal protoplast (radius, 100 microm) was investigated quantitatively by amperometric measurements using microelectrodes. Under light irradiation (25 kLx) in the presence of 1.00 mM BQ, a single protoplast consumed BQ by (2.9 +/- 0.2) x 10(-13) mol/s and generated p-hydroquinone (QH2) by (2.7 +/- 0.3) x 10(-13) mol/s, suggesting that BQ was quantitatively reduced to QH2 via the intracellular photosynthetic electron-transport chain. The generation of QH2 increased with light intensity and with concentration of BQ added to the outside solution but became saturated when the light intensity was above 15 kLx or the BQ concentration was higher than 0.75 mM. The addition of 3-(3, 4-dichlorophenyl)-1,1-dimethylurea, a photosynthetic electron-transport inhibitor, decreased the generation of QH2 upon light irradiation, suggesting that BQ accepts electrons from a site in the photosynthetic electron-transport chain after the photosystem II site. The presence of 1.00 mM BQ increased the generation of photosynthetic oxygen by approximately (2.6 +/- 1.0) x 10(-13) mol/s, which was approximately 1.5-2 times larger than that expected from the consumption of BQ. The electrons produced by the additional generation of oxygen is used to reduce intracellular species as well as to reduce BQ.