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.

Evolutionary aspects of variant types of rat mitochondrial DNA'S.

Mitochondrial DNA's (mtDNAs) were prepared from various kinds of individual Norway rats, Rattus norvegicus, and from three types of individual black rats, Rattus rattus, (Asian type, Ceylon type, and Oceanian type). Intra- and interspecies divergence of their mtDNA sequences were calculated based on changes in restriction endonuclease cleavage sites. The extent of intraspecies divergence of black rats (about 8%) is much larger than that of Norway rats (1%) and the mtDNA of Asian-type black rats resembles the mtDNA of Norway rats more closely than it resembles the mtDNA of other types of black rats. These results strongly suggest that during the course of intraspecies differentiation of black rats, probably long after the separation of the three types of black rats, some Asian-type black rats were isolated sexually and formed a new species, Norway rats. On the basis of our observations we propose a hypothetical process to explain the evolution of animal mtDNA.

Geographical distribution of twelve transferrin alleles in black rats of Asia and Oceania.

About 450 black rats (Rattus rattus) were collected from 25 localities in Asia and Oceania. Their serum transferrins were analyzed by a newly developed thin layer acrylamide gel electrophoresis accompanied with acrinol pretreatment, exhibiting 12 transferrin bands. Generally, Asian type rats (2N=42) had fast-moving transferrins (R-series), Ceylon type (2N=40) moderately moving ones and Oceanian type (2N=38) slowly moving ones (C-series). Exceptionally, in northern India and Pakistan all Asian-type rats had C-series Tf. The possibility that divergence of R-series Tf and C-series Tf had proceeded the karyotypic differentiation from 42 to 38 is proposed. In combination with the previous molecular data, the time of the divergence is roughly estimated between the order of a million years and ten thousand years.

Genetic Differentiation among Karyotypic Forms of the Black Rat, RATTUS RATTUS.

The black rat, Rattus rattus, consists of five karyotypic forms-2n = 42 (high C-banding); 2n = 42 (low C-banding); 2n = 40; 2n = 38; 2n = 42 Mauritius. Here, we use isozyme electrophoresis and microcomplement fixation to elucidate the genetic distance and phylogenetic relationship among each of the various karyotypic forms of R. rattus and R. norvegicus . The results show that (1) the 2n = 42 Mauritius black rat (2n = 42Mau) is genetically very similar to the 2n = 38 form, suggesting that this island population has undergone very rapid chromosomal evolution; (2) the 2n = 40 form from the highlands of Sri Lanka is genetically distinct from the 2n = 38 form from the lowlands; the genetic difference is probably insufficient, however, to prevent future introgression; (3) the level of genetic differentiation occurring between the 2n = 42 forms on the one hand and the 2n = 38, 2n = 40 and 2n = 42 Mau forms on the other support the hybrid incompatability data in suggesting that the two groups are either full species or incipient species; (4) in contrast to data from amino acid composition of transferrin and from restriction endonuclease digests of mtDNA, the present data suggest that the various karyotypic forms of R. rattus are phylogenetically more closely related to each other than any is to R. norvegicus, and that they are related by a series 2n = 42 --> 2n = 40 --> 2n = 38; (5) the R. rattus/R. norvegicus divergence occurred 2-8 million years ago, whereas the various chromosomal forms of R. rattus diverged over the last 4 million years.

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.

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.

Chromosome alteration and the development of tumors. XXIII. Banding karyotype analyses of methylcholanthrene-induced tumors in the Indian spiny mouse, Mus platythrix, with special regard to the anomalies of chromosomes with nucleolar organizer regions.

In the Indian spiny mouse, Mus platythrix (2n = 26), six tumors were induced by 3-methylcholanthrene, and their karyotypes were analyzed in the primary state by G-banding. The chromosome numbers of these tumors were widely distributed ranging from diploid to tetraploid, but the frequency of cells exhibiting diploidy was the highest. Among these cells, the frequency of the cells with a normal diploid karyotype was only 27%, but the remaining cells (73%) showed pseudo- or near-diploid karyotypes. Although several numerical and structural anomalies of the chromosomes were observed in these tumor cells, centric fusion and translocation was most commonly seen, and that of trisomy and monosomy ranked second. Among 13 chromosome pairs, higher frequencies of chromosome anomalies were observed in the chromosomes No. 5, 8, and 12. Anomalies of the other autosomes were related primarily to centric fusion with chromosomes No, 5, 8, or 12, those of the X chromosome were mainly numerical changes. Taking into account the nucleolar organizer regions (NORs), which always occurred in chromosome pairs No. 5, 8 and 12 in this species, a possible relationship between the anomalies of those chromosomes containing NORs and the malignant transformation of cells is proposed.

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.

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.

Frequency of sister-chromatid exchanges depending on the amount of 5-bromodeoxyuridine incorporated into parental DNA.

Chinese hamster D-6 cells were grown for two cell cycles. The effect of 5-bromodeoxyuridine (BrdU) on the frequencies of sister-chromatid exchanges (SCEs) in these cells was investigated by the BrdU-labeling method. A low concentration (5 microM) of BrdU was inoculated in the first cell cycle for SCE counting. When excess concentrations (100-1000 microM) of BrdU were added subsequently in the second cell cycle, a 1-2-fold increase of SCE frequencies was observed. When excess thymidine (dT) (100-1000 microM) was supplied instead of BrdU, the incidence of SCE also increased. When cells were exposed to high concentrations (50-200 microM) of BrdU in the first cell cycle, a 1-4-fold increase in SCE frequencies was observed. This incidence of SCE was largely dependent on the concentration of BrdU and dT used in the second cell cycle. These results suggest that efficient SCE induction by BrdU is related to the BrdU residue incorporated into parental DNA strands.

Experimental breeding and cytogenetics of the soft-furred rat, Millardia meltada.

The soft-furred rat, Millardia meltada, has bred successfully in the laboratory for 10 generations of brother x sister mating. It is half way in size between the rat and the mouse, and is gentle and easy to handle. Thin body hair and incisor teeth are present in the newborn. Some individuals have low blood pressure. Chromosome number (2n) in 50. It differs in several characteristics from the rat and mouse, and could become a useful experimental animal in biomedical research.

[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

[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.

[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.

[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.

[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.