Evaluation of inosin-5'-monophosphate dehydrogenase activity during maintenance therapy with tacrolimus.

OBJECTIVES: The aim of this study was to identify a target range for inosin-5'-monophosphate dehydrogenase (IMPDH) activity in maintenance therapy with tacrolimus (TCL), and to apply the measurement of IMPDH activity to the therapeutic drug monitoring for mycophenolate mofetil (MMF). METHODS: Eleven patients with renal transplants and 10 healthy volunteers were investigated. All patients were treated with a combination of TCL, steroid and MMF for 2 months after transplantation, and were in stable and good condition. IMPDH activity was determined indirectly by measuring xanthosine 5'-monophophate in cell lysates supplemented with IMP and beta-nicotine adenine dinucleotide using an high-performance liquid chromatography (HPLC) method. RESULTS: The within-run reproducibility of the assay was excellent, with relative standard deviation (RSD) values of 0.41-4.08%. The mean differences between the spiked concentrations of xanthosine 5'-monophophate and their real values (mean relative errors; MREs) were within a range of 2.66-8.89%, showing good accuracy. The interday RSD values were 1.51-6.12% and MREs ranged from 2.10% to 8.89%. Cell lysates showed a 5-6 nmol/L IC(50) mycophenolic acid (MPA) concentration. TCL, cyclosporine and prednisolone did not affect IMPDH activity. The peak MPA concentration was achieved at 1 h after dosing. IMPDH activity decreased to 75% and 67% at 1 and 2 h after dosing respectively. Therefore, the inhibition rates of MPA against IMPDH activity may be adequate at 25-40% in TCL maintenance therapy. CONCLUSION: Inosin-5'-monophosphate dehydrogenase activity in cell lysates could be reliably determined by HPLC. A 25-40% inhibition of IMPDH activity may be an appropriate range for preventing rejection with MPF but this requires further validation using larger studies with harder outcomes such as rejection episodes.

Changes in serum hypoxanthine levels by exercise in obese subjects.

To study on effect of obesity on changes in serum hypoxanthine with exercise, exercise stress testing with treadmill was performed on 7 obese subjects (body mass index [BMI], 30.6 +/- 3.2 kg/m(2)) and 16 healthy volunteers (BMI, 21.5 +/- 2.10 kg/m(2)). Expiratory gas analysis during exercise showed that peak Vo(2) was significantly lower in the obese group than in the control group (28.1 +/- 4.0 v 37.1 +/- 4.7 mL/kg/min; P <.001). Furthermore, the obese group had lower anaerobic threshold (AT) values (P <.005), respiratory quotient at AT (P =.003), and exercise capacity reserve (P =.002) than the control group. Baseline serum hypoxanthine levels were significantly higher in the obese group than in the control group (3.46 +/- 3.70 v 1.23 +/- 1.16 micromol/L; P <.05). Exercise induced a pronounced increase in serum hypoxanthine level in the obese group compared with the control group (10.65 +/- 6.81 v 43.86 +/- 4.56 micromol/L; P <.01). Serum levels of uric acid before and after load were also higher in the obese group than in the control group (404 +/- 43 v 302 +/- 77 micromol/L; P <.005). A pronounced increase in hypoxanthine with exercise may result in organ damage caused by free radicals, and intermittent training from mild intensity may be less hazardous for exercise treatment of obesity.

[Susceptibilities of bacteria isolated from patients with respiratory infectious diseases to antibiotics (1995)].

The bacteria isolated from the patients with lower respiratory tract infections were collected by institutions located throughout Japan, since 1981. Ikemoto et al. have been investigating susceptibilities of these isolates to various antibacterial agents and antibiotics, and characteristics of the patients and isolates from them each year. Results obtained from these investigations are discussed. In 23 institutions around the entire Japan, 567 strains of presumably etiological bacteria were isolated mainly from the sputa of 459 patients with lower respiratory tract infections during the period from October 1995 to September 1996. MICs of various antibacterial agents and antibiotics were determined against 74 strains of Staphylococcus aureus, 82 strains of Streptococcus pneumoniae, 104 strains of Haemophilus influenzae, 85 strains of Pseudomonas aeruginosa (non-mucoid strains), 18 strains of Pseudomonas aeruginosa (mucoid strains), 52 strains of Moraxella subgenus Branhamella catarrhalis, 25 strains of Klebsiella pneumoniae etc., and the drug susceptibilities of these strains were assessed except for those strains that died during transportation. 1) S. aureus. S. aureus strains for which MICs of oxacillin (MPIPC) were higher than 4 micrograms/ml (methicillin-resistant S. aureus) accounted for 52.7%. Arbekacin (ABK) showed the most highest activity against S. aureus with MIC80 of 0.5 micrograms/ml. Vancomycin (VCM) showed the next highest activity with MIC80 of 1 microgram/ml. These drugs showed the high activities against MRSA with MIC80S of 1 microgram/ml. 2) S. pneumoniae. Most of drugs tested showed potent activities against S. pneumoniae. Imipenem (IPM) and panipenem (PAPM), carbapenems, showed the most potent activity with MIC80S of 0.063 microgram/ml. Cefotaxime (CTX), cefmenoxime (CMX) and cefpirome (CPR) of cephems showed the next most potent activities with MIC80S of 0.25 microgram/ml. Erythromycin (EM) and clindamycin (CLDM) showed low activities with MIC80S 128 micrograms/ml or high. Among these strains, however, 48.8% and 65.9% of respective strains were quite toward sensitive these agents with MICs of 0.063 microgram/ml. 3) H. influenzae. The activities of all drugs were potent against H. influenzae test with all MICs at 4 micrograms/ml or below. Cefotiam (CTM), CMX, cefditoren (CDTR) and ofloxacin (OFLX) showed the most potent activity with MIC90S to 0.063 microgram/ml. 4) P. aeruginosa. (mucoid strains) IPM and tobramycin (TOB) showed the most potent activity against P. aeruginosa (mucoid strains) with MIC80S of 1 microgram/ml. Ceftazidime (CAZ), cefsulodin (CFS) and carumonam (CRMN) showed next potent activity, with MIC80S of 2 micrograms/ml. The MIC80S of the other drugs ranged from 4 micrograms/ml to 32 micrograms/ml. 5) P. aeruginosa (non-mucoid strains). TOB and ciprofloxacin (CPFX) showed the most potent activities against P. aeruginosa (non-mucoid strains) with MIC80S of 1 microgram/ml. The MIC80 of ampicillin (ABPC) was 128 micrograms/ml in 1994, it was 16 micrograms/ml in 1995. 6) K. pneumoniae. All drugs except ABPC were active against K. pneumoniae. CPR and CRMN showed the most potent activities against K. pneumoniae with MIC80S of 0.063 microgram/ml. The MIC80S of the other drugs ranged from 0.125 microgram/ml to 2 micrograms/ml. 7) M. (B.) catarrhalis. Against M. (B.) catarrhalis, all the drugs showed good activities with MIC80S at 4 micrograms/ml or below. And MICs of all strains were 8 micrograms/ml or below. IPM, OFLX and minocycline (MINO) showed the most potent activity with MIC80S of 0.063 microgram/ml. Also, we investigated year to year changes in the characteristics of patients, their respiratory infectious diseases, and the etiology. Patients' backgrounds were examine for 567 isolates from 459 cases. The examination of age distribution found that the proportion of patients with ages over 60 years was 66.3% of all the patients showing a slight increase over that in 1994. Proportion of differe

Combined analysis of expression of c-erbB-2, Ki-67 antigen, and tenascin provides a better prognostic indicator of carcinoma of the papilla of Vater.

Expression of the molecular biological factors (MBFs) c-erbB-2, Ki-67 antigen, and tenascin (TN) was assessed immunohistochemically in specimens from patients who had undergone surgery for carcinoma of the papilla of Vater. The MBFs were then analyzed by histological factors (v, d, panc, n, Stage), which have been demonstrated to be outcome predictors, and by patient outcome. None of the MBFs showed any significant correlation with the histological factors. There were significant differences (p < 0.05) in the expression of c-erbB-2, Ki-67 antigen, and TN between patients who survived >5 years and those who survived <5 years. The patients with greater expression of c-erbB-2, Ki-67 antigen, and TN had a poor prognosis, whereas those with less expression had a good prognosis. They were therefore considered independent predictors of outcome for carcinoma of the papilla of Vater. Combined analysis of both histological factors and MBFs was also performed, with the result that the combined analysis of MBFs yielded a better prediction of outcome in carcinoma of the papilla of Vater than analysis of either one histological factor or MBF.

Biological monitoring of workers exposed to N,N-dimethylformamide by determination of the urinary metabolites, N-methylformamide and N-acetyl-S-(N-methylcarbamoyl) cysteine.

Biological monitoring of workers exposed to N,N-dimethylformamide (DMF) was carried out by determination of the urinary metabolites, N-methylformamide (MF, mainly from N-hydroxymethylformamide) and N-acetyl-S-(N-methylcarbamoyl)cysteine (AMCC), which were derived from two different routes of metabolism of the solvent. The urinary levels of MF increased rapidly at the start of the work shift, and decreased almost to zero within 24 h after the beginning of the last exposure. The highest level was found between the end of the afternoon shift and bedtime. AMCC levels remained constant over the consecutive work days and increased after the cessation of exposure, with the peak concentration being observed at 16-40 h after the cessation of exposure. AMCC levels at the beginning of the next morning shift were closely correlated with personal exposure levels of DMF in air, although the correlation of MF and DMF in air was highest in the urine at the end of the shift. Hence urinary AMCC represents an index of the average exposure during several preceding work days and may indicate the internal dose. By contrast, MF represents an index of daily exposure.

[Study on combined of auranofin, salazosulfapyridine and methotrexate in rheumatoid arthritis].

Patients with rheumatoid arthritis who did not respond sufficiently to auranofin therapy were divided into three groups to be continued on auranofin monotherapy (1), to be on combination therapy of auranofin and salazosulfapyridine (2) and to be given methotrexate concomitantly with auranofin (3), and clinical responses were compared in a prospective manner. Patients on combination therapy either with salazosulfapyridine or with methotrexate demonstrated higher improvement than those on continued auranofin monotherapy. No unknown side effects associated with monotherapy of any of the three compounds were seen in any of the three groups. The study suggests usefulness of combination therapy either with methotrexate or with salazosulfapyridine for patients who failed to respond sufficiently to auranofin.

Instability of pleomorphic tubulin paracrystals artificially induced by Vinca alkaloids in tissue-cultured cells.

The processes of tubulin paracrystal induction in Chinese hamster ovary cells treated with a Vinca alkaloid, ie, vinblastine or vincristine, and treated simultaneously with one of the Vinca alkaloids and colcemid or colchicine were followed by four different microscopic techniques, in particular by tubulin-immunofluorescence. Vinca alkaloid alone, in lower concentrations, induced basically tactoid or needle-shaped (N-shaped) paracrystals. However, the formation of crystalloid was greatly enhanced by increasing the concentration of Vinca alkaloid. Square or barrel-shaped (S-shaped) and hexagonal paracrystals were also commonly induced by simultaneous treatment with a Vinca alkaloid and colcemid or colchicine. Large rectangular paracrystals often displayed fibrillar or lamellar fine structures which ran perpendicular to the long axis but tended to cleave into fragments by spontaneous splitting. Electron micrographs revealed the fine structure of crystalloids to be aggregates of numerous filaments. The growth of paracrystals, particularly N-shaped crystals, was markedly inhibited when cells were exposed to drug(s) at a low temperature (4 degrees C). We confirmed that both N- and S-shaped paracrystals dissociated rapidly after the culture medium was replaced with fresh, drug-free medium. Glutaraldehyde-fixed paracrystals treated with RNase solution were stained with acridine orange, showing a weak orange color. Possible factors involved in the assembly and disassembly of tubulin paracrystals are discussed.

Dermatoglyphic development on the volar pads of rats with chromosome abnormalities.

Dermatoglyphic abnormalities are often observed in patients with chromosome aberrations, but no similar observations have been made in animals. In the present study, palmar dermatoglyphics were examined in 4 rats with chromosome anomalies. Reciprocal translocations were induced by gamma-irradiation; the animals used were obtained from among offspring with abnormal karyotypes that were derived from the original mutant rats. As the epidermal surface of the volar pad of the rat is flat, dermatoglyphic characteristics were observed on the dermal surface following staining with toluidine blue. Unusual ridge configurations were found in some of them, suggesting that dermatoglyphic development in the rat reflects, to some extent, an abnormal chromosome constitution.

Preferential clustering of viral DNA sequences at or near the site of chromosomal rearrangement in fowl adenovirus type 1 DNA-transformed cell lines.

All six transformants obtained by inoculating fowl adenovirus type 1 (CELO virus) DNA or its fragments into a rat cell line of normal karyotype had more than 50 copy-equivalents of viral DNA sequences. Each of the transformants had almost all if not all of these viral DNA sequences clustered on a marker chromosome(s). Although the marker chromosome(s) differed from one cell line to another, viral DNA sequences preferentially clustered in or near the achromatic (or light-stained) region of the G-banded marker chromosomes where chromosomal rearrangement or translocation occurred. These results indicate that no particular chromosome is required to act as the integration site of viral DNA for the transformation of cells, but chromosomal rearrangement at or near the cluster of viral DNA sequences might contribute to the transformation.

Inheritance of dermatoglyphic configurations in the rat.

Inheritance of dermatoglyphic configurations was studied on the palmar III interdigital pad of the rat (Rattus norvegicus). Four rats from each of the WKS and ACI/N inbred strains were mated with each other, and 54 F1 and 88 F2 hybrids were obtained. In addition, 52 offspring were produced by backcross mating between F1 hybrids and WKS and ACI/N parents. Whorls were the predominant pattern in the WKS strain and triradial patterns characterized the ACI/N strains. The F1 hybrids showed a wide range of pattern types, consisting of whorls, loops, cusps, arches, and triradial patterns. These patterns were intermediate in size between the two inbred strains. The F2 hybrids presented a distribution of patterns with a similar range as the F1, but the frequencies of some types were different. Patterns in the offspring of each backcross demonstrated a slight shift towards the characteristic pattern of the parental inbred strain. No sex difference was observed. Generally, the bilateral differences were striking, with a radial direction predominating on the left palm, and an ulnar one on the right palm, respectively. This study suggests that the dermal patterns are genetically determined, but also are influenced by environmental factors, especially in the hybrids.

[Double channel chemotherapy with intra-arterial infusion of DDP and concurrent intra-venous infusion of sodium thiosulfate (STS) in bladder cancer].

Ten patients with bladder cancer were treated with double channel chemotherapy which included intra-arterial infusion of DDP and concurrent intra-venous infusion of STS, a neutralizing agent against DDP. The method allows the administration of a relatively high dose of DDP to localized malignant tumors by protection from systemic cytotoxicity of DDP by STS. DDP in a dose of 100-150 mg/m2 of body surface was infused either to the internal iliac artery at a portion close to the superior vesical artery or to the aortic bifurcation by the Seldinger technique over 30 minutes, and infusion of 20 g STS to the peripheral vein was simultaneously started and continued for 2-3 hours. One patient achieved a complete response and 2 patients achieved a partial response. The response rate was 30%. Histopathologically, 4 out of 8 patients showed an effect of 2 grades or more by Oboshi-Shimosato's criteria. Invasive or hypervascular tumors seem to be more responsive to the present treatment than superficial or hypovascular ones. Side effects were relatively mild inspite of a large dose of DDP. It should be noted that concurrent STS infusion efficiently counteracts the renal toxicity of DDP.

Malignant transformation of Bloom syndrome B-lymphoblastoid cell lines by carcinogens.

Three types of Bloom syndrome B-lymphoblastoid cell lines, as well as one derived from a normal person, treated with 4-nitroquinoline-N-oxide and N-methyl-N'-nitro-N-nitrosoguanidine (0.3 micrograms/ml for 24 hr), were studied for tumorigenicity in nude mice, colony formation in soft agar, cytogenetic changes, and immunoglobulin markers. When normal and Bloom syndrome cells with normal sister chromatid exchange (SCE) levels and karyotypes (type I) were treated with carcinogens, no significant changes occurred in the immunoglobulin profile and karyotype, only rare colony formation was seen, and no tumors were produced. In contrast, when Bloom syndrome cells with high SCE levels (type II with normal karyotype and type III with an abnormal karyotype) were treated with carcinogens, tumors were produced in 22 of 53 nude mice injected; a high rate of colony formation in soft agar was seen; the cells exhibited virtual loss of immunoglobulin markers; and structural changes in chromosomes 1, 2, 3, 4, 5, 7, 11, 14, and 15 were found in the tumors in addition to the original chromosome abnormalities present in the injected cells. It appears that Bloom syndrome B-lymphoblastoid cell lines with high levels of SCE are highly susceptible to the action of carcinogens, as evidenced by tumor formation in nude mice and colony formation in agar. Apparently, the carcinogens were capable of transforming only those cells that had a critical level of SCE (approximately 140 per cell) and not those with only mildly increased levels (approximately 13 per cell).

[Development of technics for observation of chromosomes by the differential staining of sister chromatids, high resolution banding and the in situ molecular hybridization].

Cytogenetical technique newly developed for the observation of chromosomes, such as differential staining of sister-chromatids the high-resolution banding and in situ molecular hybridization are introduced in this chapter. In the differential sister-chromatid staining, acricine-orange, BUdR and FPG techniques are described. In the high resolution banding, amethopterin, BUdR and ethidium methods are introduced. RNA/DNA and DNA/DNA in situ hybridization techniques are also included.

[Technique for the karyotype analysis].

Techniques for the karyotype analysis in the man and some laboratory animals are described in the present chapter. The international systems for the representation of the standard karyotypes in these materials due to the conventional and the several banding stainings are also introduced.

Inhibition of bromodeoxyuridine-associated sister chromatid exchanges in Bloom's syndrome cells with cycloheximide.

Effects of cycloheximide (CH) and deoxycytidine (dC) on the frequency of sister chromatid exchanges (SCEs) in normal and Bloom's syndrome (BS) cells labeled with bromodeoxyuridine (BrdU) during first, second, and third cell cycles were evaluated using endomitotic and three-way differentiation analyses. When CH at 0.2 and 2.0 ng/ml was added to normal and BS cultures of BrdU-labeled endomitoses, the rate of single SCEs was significantly decreased in BS cells, though the rate of reduction in single SCEs was slight in normal cells. No significant change was detected in the twin SCE rate. In BS cells, treatment with CH at 0.2 and 2.0 ng/ml produced significant reductions in SCEs in both the second (SCE2) and third (SCE3) cell cycles, sometimes reaching the normal level. Treatment with dC at 13 and 26 micrograms/ml resulted in almost no significant changes in rates of SCE during first, second, and third cell cycles. When CH was added to BrdU-labeled normal and BS cell cultures, the cell growth rates improved from 35% to 70% over the control level in the BS cells, though in normal cells, the addition of CH resulted in a close-dependent lower cell growth rate. Deoxycytidine did not noticeably affect the cell growth rates in BrdU-labeled normal and BS cultures. The finding that the reduction of BrdU-induced SCEs in BS is paralleled by cell growth improvement is of special interest.

[Cytogenetic techniques for the observation of chromosomes from the cultured cells of the blood and amniotic fluid].

Cytogenetical techniques of the observation of chromosomes in mammals (human) by the cultivation of the peripheral blood cells and also cells suspending in the amniotic fluid are described in this paper. In the blood cell cultivations, the common technique by use of the leukocytes and also a small amount of the whole blood are introduced. The culture methods of the cells including in the amniotic fluid are also described with the technique of the chromosome preparations.