Comparison of activities of enzymes related to energy metabolism in peripheral leukocytes and livers between Holstein dairy cows and ICR mice.

Activities of enzymes related to energy metabolism and isoenzyme patterns of lactate dehydrogenase (LDH) were determined in peripheral leukocytes and livers of Holstein dairy cows and Institute of Cancer Research (ICR) mice. In dairy cow liver, activities of enzymes in glycolysis, malate-aspartate shuttle and lipogenesis were lower, but activities of glucose-6-phosphatase in gluconeogenesis were higher than those in mouse liver. Glucokinase activities were below detection limit in leukocytes and liver of the cows. Dairy cow leukocytes and liver showed the isoenzyme patterns with dominance of LDH-1, -2 and-3, whereas mouse leukocytes and liver showed that LDH-5 was dominant. The LDH isoenzyme patterns were very similar between leukocytes and liver in each animal species. Some enzymes in leukocytes may reflect those enzymes activities in liver and be a useful indicator for energy metabolism in animals.

Changes in activities of enzymes related to energy metabolism in peripheral leukocytes of fattening steers.

Glucose, triglyceride, cholesterol and immunoreactive insulin (IRI) concentrations, some enzyme activities in plasma, and activities of enzymes related to energy metabolism in peripheral leukocytes were measured in fattening Japanese Black Wagyu x Holstein steers fed on different diets at 8, 12, 16, 20 and 24 months of age. The plasma IRI concentrations at 20 and 24 months of age were significantly higher than those at 8 months of age. Activities of hexokinase (HK), glucose-6-phosphate dehydrogenase (G6PD), aspartate aminotransferase (AST), and malate dehydrogenase (MDH) in cytosolic fractions, and glutamate dehydrogenase (GLDH), MDH and AST in mitochondrial fractions in peripheral leukocytes of steers at 24 months of age were significantly higher than those at 8 months. Increasing plasma insulin concentration was considered to induce acceleration of glucose utilization in leukocytes of fattening steers. The cytosolic ratio of MDH/lactate dehydrogenase (LDH) activity in leukocytes increased significantly in the fattening process and was considered to be a useful indicator for evaluating changes in energy metabolism in steers.

The effect of measurement error on the dose-response curve.

In epidemiological studies for an environmental risk assessment, doses are often observed with errors. However, they have received little attention in data analysis. This paper studies the effect of measurement errors on the observed dose-response curve. Under the assumptions of the monotone likelihood ratio on errors and a monotone increasing dose-response curve, it is verified that the slope of the observed dose-response curve is likely to be gentler than the true one. The observed variance of responses are not so homogeneous as to be expected under models without errors. The estimation of parameters in a hockey-stick type dose-response curve with a threshold is considered on line of the maximum likelihood method for a functional relationship model. Numerical examples adaptable to the data in a 1986 study of the effect of air pollution that was conducted in Japan are also presented. The proposed model is proved to be suitable to the data in the example cited in this paper.

Notes on the use of historical controls.

The principles and methods of incorporating historical controls in four cases (C1, stable control; C2, rare occurrence of responses; C3, small group size; C4, historical control as a reference) are discussed. Two points are emphasized: one is that the historical control should be regarded as a given condition and the other is that the historical control should be used conservatively. Incorporating historical controls is recommended only when it is advantageous under the conditional evaluation of the performance and even in the conservative use of controls. For case C1, adjusting the critical value for the Cochran-Armitage trend test is proposed. For case C2, a modified conditional trend test proposed by Yanagawa et al. is appreciated as a proper procedure. For case C3, a conservative use of interblock information is discussed. The incorporation of the historical control is not recommended for case C4.

Statistical analysis of data in mutagenicity assays: rodent micronucleus assay.

To evaluate chemical safety, many kinds of short-term mutagenicity assays are performed together with long-term assays in animals. Rationales and methodology for these assays have been well discussed and documented. No statistical method, however, has been singled out as the method of choice for the evaluation of mutagenicity assay data, although a number of reports on statistical methods to evaluate such data have been published. Among the mutagenicity assays, the micronucleus assay using mouse bone marrow erythropoietic cells have been widely used to assess cytogenetic activities of test chemicals. A statistical evaluation procedure for this assay is proposed herein, combining the use of historical control data and dose-response relationships. The probability of type I errors and the power of this method are compared with those of some other conventional methods by Monte Carlo simulation.

A procedure for data analysis of the rodent micronucleus test involving a historical control.

No standard procedure of data analysis for rodent micronucleus tests involving historical controls has been established. In the present paper, under the presumption that the distribution of the historical control is stable and reliable, a procedure with three statistical steps is proposed to analyze the frequency of micronucleated polychromatic erythrocytes (MNPCEs). In the first step, the frequencies of MNPCEs in negative and positive control groups of a current experiment of the micronucleus test are compared with the distribution of historical negative and positive controls to examine the technical validity of the current experiment. In the second step, the frequency of MNPCEs in each treatment group is compared with the distribution of the historical negative control. In the third step, the dose-response relation is tested with the Cochran-Armitage trend test. A Monte Carlo stimulation study shows that the power of this procedure is acceptable and also this procedure is robust. An application of this procedure on real data reveals that it is effective in detecting clastogenic chemicals when the probability of a type I error is nearly .01.

Report and summary of the major conclusions from statistics in genotoxicity testing working group from the International Workshop on Genotoxicity Test Procedures (IWGTP), March 1999.

A working group of five statisticians experienced in the use of statistical methods in mutagenicity reviewed aspects of the statistical analysis of genotoxicity test procedures. Issues discussed included methods for integrating biological importance and statistical significance, the relationship of the experimental unit to the experimental design, and the impact of new developments in statistics and computing. Three major recommendations were made relating to the need for: (1) the effective use of statistical advice in designing interlaboratory and intralaboratory investigations; (2) the development of appropriate experimental designs for new assays; and (3) education and training in the use of statistical methodology in mutagenicity testing. Environ. Mol. Mutagen. 35:260-263, 2000 Published 2000 Wiley-Liss, Inc.

An amino acid from Solorina crocea.

A new amino acid, named solorinine, isolated from the lichen, Solorina crocea was determined by a single-crystal X-ray diffraction analysis. NMR data are also reported.

Long-term observations of two siblings with Lafora disease treated with zonisamide.

We have reported long-term clinical follow-up for two siblings with Lafora disease, a brother and sister, one of whom autopsied. Both siblings had repeated attacks of severe myoclonus, tonic and tonic-clonic convulsions, and intractable status epilepticus. The addition of orally administered zonisamide brought about striking effective seizure control for about 12-14 years in both patients, relieving not only myoclonus and generalized tonic-clonic seizures but also intractable status epilepticus.

Effects of age on communication skill levels and prevalence of maladaptive behaviors in autistic and mentally retarded children.

This study investigated the effect of age on communication and maladaptive behavior in autistic and mentally retarded children. Forty-seven autistic and 128 mentally retarded children in a special school served as subjects. The cross-sectional method was used to compare junior and senior groups, and ratings on communication skills and maladaptive behaviors were obtained from teachers. We found that the skills of comprehension and conversation in autistic children improved significantly with age, and speech improved somewhat. In spite of this improvement in communication skills, maladaptive behaviors in the autistic children other than hyperactivity did not change significantly with age. Withdrawal improved significantly with age in the mentally retarded children but not in the autistic children.

Effects of age on adaptive behavior levels and academic skill levels in autistic and mentally retarded children.

This study investigated the effect of age on adaptive behavior and academic skill in autistic and mentally retarded children. Subjects were 47 autistic and 128 mentally retarded children from a special school. Cross-sectional comparisons were made between junior and senior groups using ratings obtained from teachers on adaptive behaviors and academic skills. We found that the levels of toilet training, eating skills, participation in group activities, and self-control in the autistic children improved significantly with age. The skills of number concepts in the autistic children also improved with age. However, these adaptive and academic levels were in general significantly lower than those of the mentally retarded children. The levels of initiative did not improve significantly in either the autistic or the mentally retarded children, and they were significantly lower in the autistic children. The implications of these findings in the context of our previous study on the changes of communication and maladaptive behaviors with age in the autistic children are discussed.

Recommendations for statistical designs of in vivo mutagenicity tests with regard to subsequent statistical analysis.

A workshop was held on September 13 and 14, 1993, at the GSF, Neuherberg, Germany, to start a discussion of experimental design and statistical analysis issues for three in vivo mutagenicity test systems, the micronucleus test in mouse bone marrow/peripheral blood, the chromosomal aberration tests in mouse bone marrow/differentiating spermatogonia, and the mouse dominant lethal test. The discussion has now come to conclusions which we would like to make generally known. Rather than dwell upon specific statistical tests which could be used for data analysis, serious consideration was given to test design. However, the test design, its power of detecting a given increase of adverse effects and the test statistics are interrelated. Detailed analyses of historical negative control data led to important recommendations for each test system. Concerning the statistical sensitivity parameters, a type I error of 0.05 (one tailed), a type II error of 0.20 and a dose related increase of twice the background (negative control) frequencies were generally adopted. It was recommended that sufficient observations (cells, implants) be planned for each analysis unit (animal) so that at least one adverse outcome (micronucleus, aberrant cell, dead implant) would likely be observed. The treated animal was the smallest unit of analysis allowed. On the basis of these general consideration the sample size was determined for each of the three assays. A minimum of 2000 immature erythrocytes/animal should be scored for micronuclei from each of at least 4 animals in each comparison group in the micronucleus assays. A minimum of 200 cells should be scored for chromosomal aberrations from each of at least 5 animals in each comparison group in the aberration assays. In the dominant lethal test, a minimum of 400 implants (40-50 pregnant females) are required per dose group for each mating period. The analysis unit for the dominant lethal test would be the treated male unless the background frequency of dead implants (DI) is so low that multiple males would need to be integrated to meet the minimum observation of one adverse outcome (DI) per analysis unit. A three-step strategy of data analysis was proposed for the cytogenetic assays. Use of negative historical controls was allowed in certain circumstances for interpretation of results from micronucleus tests and chromosomal aberration tests.

Evaluation of seven alternative assays on the main ingredients in cosmetics as predictors of Draize eye irritation scores.

This study evaluates seven alternative assays carried out on the main ingredients in cosmetics to determine which battery is the best set and what is the best predictor of the maximal Draize rabbit eye irritation scores (MDESs). The assays consisted of the maximal primary Draize rabbit skin irritation scores (MDSSs), a cytotoxicity test on neutral red uptake using Chinese hamster lung cells (NR-EC(50)), a cytotoxicity test on MTT using normal skin fibroblasts (MTT-EC(50)), the hen's egg test-chorioallantoic membrane test using fertile chicken eggs (HET-CAM), a haemolysis test using red blood cells from Wistar rats (HC(50)), a protein denaturation test using haemoglobin from bovine (HDR), and pH. We tested 10% solutions of 24 tested chemicals, that is, 20 surfactants, three solvents and formaldehyde, to select from the assays a best set for prediction (x) and to obtain the best predictor [f(x)] based on the prediction sum of squares criterion. The selected set consisted of NR-EC(50) and HDR, and the resultant best predictor was f(x) = 74.0 - 29.52 log(NR-EC(50)) + 0.87 HDR. This predictor achieved a high score of 89.6% of the contribution ratio. For two of the 24 test chemicals, an inconsistency occurred in the criticality of corneal damage between the observed and predicted Draize eye scores, although this is not considered to affect significantly the overall results.

Prevalence of maladaptive behavior in retarded children as a function of IQ and age.

One hundred and twenty-eight mentally retarded children, ages 6 to 14, from a special school were assessed by teachers in terms of nine maladaptive behaviors. The results indicated that the prevalence of some maladaptive behaviors changed as a function of IQ and age, and that there were splits of such changes as IQ-dependent and as age-dependent by maladaptive behavior.

Tree-type algorithm for statistical analysis in chronic toxicity studies.

An appropriate statistical methodology in toxicity studies has been discussed over the last two decades and many statistical methods have already been proposed. Many practical problems, however, still remain unresolved and most pharmaceutical industries have been using a tree-type algorithm routinely to analyze repeated-dose toxicity study data. In considering routine use of statistical analysis in toxicological studies, standardization of statistical methodology is necessary and the decision tree has an important role. In this article, the problems, relating to tree-type algorithms are summarized. Then we propose a new tree-type algorithm, which targets quantitative data in repeated-dose studies in rodents, usually sample size per group between 10 to 20, based on the following two important principles: "using a parametric method" and "suitable for intuition of toxicologists". An example of its application to actual toxicity study data is demonstrated. The performance of this new method is also evaluated using historical data. However, it should be noted that the intention of this paper is not to make a definite solution of the decision tree. Several other alternatives can be considered. Since there is no single theoretically correct solution of tree-type algorithms, too formal a use of the decision tree is not recommended. We must not forget the exploratory nature of evaluating repeated toxicity data.

An Interlaboratory Validation Study of the Improved Transformation Assay Employing Balb/c 3T3 Cells: Results of a Collaborative Study on the Two-stage Cell Transformation Assay by the Non-genotoxic Carcinogen Study Group.

The Non-genotoxic Carcinogen Study Group of the Environmental Mutagen Society of Japan organised the first step of an interlaboratory validation study on an improved cell transformation assay employing Balb/c 3T3 A31-1-1 cells. Nineteen laboratories participated in this study. The modified transformation assay was evaluated for its responsiveness, its interlaboratory reproducibility and its transferability. In this study, a mixture of Dulbecco's modified Eagle's medium and nutrient mixture F12, supplemented with insulin-transferrin-ethanolamine-sodium selenite and 2% fetal bovine serum (FBS) was used during the period of expression of transformed foci, intead of the usual minimum essential medium with 10% FBS. 20-Methylcholanthrene (MCA) and 12-O-tetradecanoylphorbol-13-acetate (TPA) were selected as a prototype initiator and a tumour promoter, respectively. Two series of experiments were conducted. In the first series, the transformation activity of MCA was examined at various concentrations. In the absence of the promoting treatment with TPA, exposure to MCA only weakly induced transformed foci. In the presence of 0.1µg/ml TPA, all laboratories observed significant dose-dependent increases in the number of transformed foci with increasing MCA concentrations. In the second series of experiments, various concentrations of TPA were tested. In the absence of initiating treatment with MCA, exposure to TPA weakly induced transformed foci in about half of the laboratories. In the presence of 0.2µg/ml MCA, all the laboratories observed significant dose-dependent increases in the number of transformed foci with increasing TPA concentrations. The results from this study support the usefulness of this modified two-stage transformation assay with Balb/c 3T3 cells.

Os vesalianum as a cause of lateral foot pain: a familial case and its treatment.

This is a rare case of bilateral symptomatic os vesalianum in a 13-year-old girl whose mother had the same condition unilaterally. We performed osteosynthesis and bone grafting instead of a simple resection to preserve the peroneus brevis tendon, with excellent results.

[Clinical efficacy and safety of the selective oral neuraminidase inhibitor oseltamivir in treating acute influenza--placebo-controlled double-blind multicenter phase III trial].

We conducted the placebo-controlled double-blind multicenter Phase III trial of newly developed selective oral neuraminidase inhibitor, oseltamivir phosphate (Ro64-0796), in order to evaluate the efficacy and safety, when Ro64-0796 was administered orally to both type A and type B influenzavirus infected patients. Patients were randomly assigned to either Ro64-0796 75 mg twice daily group or matching placebo group for five days. A total of 316 patients (Ro64-0796 group; 154 and placebo group; 162) were recruited, and intent-to-treat infected population, which was defined as the patients that study drug was administered one or more and laboratory-confirmed influenzavirus infection was demonstrated, were 122 and 130, respectively. Ro64-0796 decreased significantly median viral titers after 72 hours (p = 0.0009. Analysis of covariance), indicating the rapid inhibition of virus replication, and duration of illness which was primary variable of efficacy, was reduced statistically significant by one day (23.3 hours) (p = 0.0216, generalized Wilcoxon test). Ro64-0796 treatment also resulted in the reduction of the fever duration and severity of clinical symptoms. Concerning the safety evaluation, the main accompanied symptoms with Ro64-0796 application were gastrointestinal disorders such as bellyache, nausea and vomiting. Most of these events were mild and allowable for the clinical use. There was no abnormal change attributable to Ro64-0796 application in the clinical laboratory tests as well as the physiological tests. Our data suggests that Ro64-0796 is useful in treating the acute influenzavirus infection.

[Efficacy and safety of the selective oral neuraminidase inhibitor oseltamivir for prophylaxis against influenza--placebo-controlled double-blind multicenter phase III trial].

We have investigated the long-term prophylactic efficacy and safety of oseltamivir phosphate (Ro64-0796), an orally bioavailable prodrug of novel, potent and selective type A and type B influenzavirus neuraminidase inhibitor, when Ro64-0796 was administered orally to the healthy volunteers. Participants older than 16 year-old were randomly assigned to either Ro64-0796 75 mg once daily group or matching placebo group for six weeks. A total of 308 participants (Placebo group; 153 and Ro64-0796 group; 155) were enrolled in this trial. The primary variable of efficacy, incidence of laboratory-confirmed influenzavirus infected subjects accompanied by both fever of 37.5 degrees C or higher and at least two influenza symptoms (group 1) were 1.3% in Ro64-0796 group in contrast with 8.5% in placebo group, inducing 85% inhibition of infection (p = 0.00323, Fisher's exact test). As secondary variable, incidence of laboratory-confirmed influenzavirus infected subjects who lack either fever (37.5 degrees C or higher) or at least two influenza symptoms (group 2) and incidence of asymptomatic infected subjects (group 3) were tend to decrease in Ro64-0796 group, and finally cumulative inhibition rate was 76% in group 1 + 2 combined (p = 0.000891. Fisher's exact test), and 63% in group 1 + 2 + 3 combined (p = 0.002150, Fisher's exact test). As for the safety evaluations, Ro64-0796 was well tolerated but was associated with gastrointestinal disorders such as nausea and vomiting which were mild and allowable for the clinical use. There was no abnormal change attributable to Ro64-0796 application in the clinical laboratory tests as well as the physiological tests. Our results demonstrate that oseltamivir is safe and effective for the prevention of influenza.

[Neuropsychological studies of two subjects after the transection of the splenium of the corpus callosum (Part 3. The tactile recognition) (author's transl)].

We made the neuropsychological tactile studies of two right-handed subjects who had undergone the transection of the splenium of the corpus callosum for the pineal operation (the teratoma by the histopathological examination). The transaction grade was 2.0 cm and 2.5 cm. In this paper we examined the tactile recognition neuropsychologically: A. Superficial sensory modalities: light touch, pain perception, temperature discriminations. B. Deep sensory modalities: vibration perception, position sense, motor sense. C. Perception of material: rough or smooth, heavy or light, hard or soft. D. Perception of size: appreciation of shape in two dimentions: circle, triangle, square, cross, rhomb, appreciation of form in three dimensions: ball, column, cone, trignonal prism, cube. E. Identification of simple objects: eraser, pencil, hair pencil, fountainpen, telephone, glasses. F. Identification of japanese letters: Kana (phonetic symbols) and Kanji (essentially nonphonetic logographic symbols representing lexical morphemes). According to the papers of Akelaitis, Van Vleuten, Goldstein, the subjects after the transection of the corpus callosum could name the objects and the card board letters placed in their right hands with their eyes closed, but they could not name in their left hand by contrast. In our subjects, the identification of simple objects and the identification of japanese letters were complete good. But they had the neuropsychological visual and auditive disorders. The result shows that the commissure fibres of the identification of the simple objects and letters of two hemispheres does not connect through the splenium of the corpus callosum.