ITPKC and CASP3 polymorphisms and risks for IVIG unresponsiveness and coronary artery lesion formation in Kawasaki disease.

Functional single-nucleotide polymorphisms (SNPs) in inositol 1,4,5-trisphosphate 3-kinase C (ITPKC) (rs28493229) and caspase-3 (CASP3) (rs113420705; formerly rs72689236) are associated with susceptibility to Kawasaki's disease (KD). To evaluate the involvement of these 2 SNPs in the risk for intravenous immunoglobulin (IVIG) unresponsiveness, we investigated 204 Japanese KD patients who received a single IVIG dose of 2 g kg(-1) (n=70) or 1 g kg(-1) daily for 2 days (n=134). The susceptibility allele of both SNPs showed a trend of overrepresentation in IVIG non-responders and, in combined analysis of these SNPs, patients with at least 1 susceptible allele at both loci had a higher risk for IVIG unresponsiveness (P=0.0014). In 335 prospectively collected KD patients who were treated with IVIG (2 g kg(-1)), this 2-locus model showed a more significant association with resistance to initial and additional IVIG (P=0.011) compared with individual SNPs. We observed a significant association when all KD patients with coronary artery lesions were analyzed with the 2-locus model (P=0.0031). Our findings strongly suggest the existence of genetic factors affecting patients' responses to treatment and the risk for cardiac complications, and provide clues toward understanding the pathophysiology of KD inflammation.

Amyloid beta inhibits ectodomain shedding of N-cadherin via down-regulation of cell-surface NMDA receptor.

Dysfunction in the synapse is recognized as an early and the primary pathological process in Alzheimer's disease (AD). N-cadherin, an essential adhesion molecule for excitatory synaptic contact, forms a complex with presenilin 1 (PS1) and beta-catenin in the synaptic membrane. N-cadherin is sequentially cleaved by ADAM10 and PS1/gamma-secretase, producing a cytoplasmic fragment, N-cadherin C-terminal fragment (Ncad/CTF2) after NMDA receptor stimulation [Marambaud P, Wen PH, Dutt A, Shioi J, Takashima A, Siman R, Robakis NK (2003) A CBP binding transcriptional repressor produced by the PS1/epsilon-cleavage of N-cadherin is inhibited by PS1 FAD mutations. Cell 114:635-645; Reiss K, Maretzky T, Ludwig A, Tousseyn T, de Strooper B, Hartmann D, Saftig P (2005) ADAM10 cleavage of N-cadherin and regulation of cell-cell adhesion and beta-catenin nuclear signalling. EMBO J 24:1762]. Ncad/CTF2 translocates to the nucleus together with beta-catenin to enhance beta-catenin nuclear signaling [Uemura K, Kihara T, Kuzuya A, Okawa K, Nishimoto T, Bito H, Ninomiya H, Sugimoto H, Kinoshita A, Shimohama S (2006a) Activity-dependent regulation of beta-catenin via epsilon-cleavage of N-cadherin. Biochem Biophys Res Commun 345:951-958]. To examine whether an impairment of N-cadherin metabolism is involved in AD pathogenesis, we investigated the effect of amyloid beta peptide (Abeta) treatment on sequential N-cadherin cleavage. Here, we demonstrate that both synthetic and cell-derived Abeta species inhibit ectodomain shedding of mouse N-cadherin. Inhibition of N-cadherin cleavage by Abeta treatment was suggested to be mediated by the enhanced endocytosis of NMDA receptor, resulting in reduced turnover of N-cadherin. Since both N-cadherin and beta-catenin are essential for synaptic plasticity, impairment of N-cadherin cleavage caused by Abeta may underlie the synapse toxicity involved in AD pathogenesis.

Developmental and transcriptional consequences of mutations in Drosophila TAF(II)60.

In vitro, the TAF(II)60 component of the TFIID complex contributes to RNA polymerase II transcription initiation by serving as a coactivator that interacts with specific activator proteins and possibly as a promoter selectivity factor that interacts with the downstream promoter element. In vivo roles for TAF(II)60 in metazoan transcription are not as clear. Here we have investigated the developmental and transcriptional requirements for TAF(II)60 by analyzing four independent Drosophila melanogaster TAF(II)60 mutants. Loss-of-function mutations in Drosophila TAF(II)60 result in lethality, indicating that TAF(II)60 provides a nonredundant function in vivo. Molecular analysis of TAF(II)60 alleles revealed that essential TAF(II)60 functions are provided by two evolutionarily conserved regions located in the N-terminal half of the protein. TAF(II)60 is required at all stages of Drosophila development, in both germ cells and somatic cells. Expression of TAF(II)60 from a transgene rescued the lethality of TAF(II)60 mutants and exposed requirements for TAF(II)60 during imaginal development, spermatogenesis, and oogenesis. Phenotypes of rescued TAF(II)60 mutant flies implicate TAF(II)60 in transcriptional mechanisms that regulate cell growth and cell fate specification and suggest that TAF(II)60 is a limiting component of the machinery that regulates the transcription of dosage-sensitive genes. Finally, TAF(II)60 plays roles in developmental regulation of gene expression that are distinct from those of other TAF(II) proteins.

Autonomic imbalance induced breakdown of long-range dependence in healthy heart rate.

OBJECTIVES: The investigation of the relation between the long-range correlation property of heart rate and autonomic balance. METHODS: An investigation of the fractal scaling properties of heart rate variability was carried out by using detrended fluctuation analysis (DFA). Eleven healthy subjects were examined for two consecutive days, which included usual daily activity, strenuous prolonged experimental exercise, and sleep. We also considered two patient groups with autonomic dysfunction characterized by selective sympathetic and parasympathetic dominance. RESULTS: Robust long-range dependence in heart rate is observed only in the state of usual daily activity, characterized by normal heart rate typical of balanced autonomic sympathetic and parasympathetic regulation. This confirms the previously postulated behavioral independence of heart rate regulation, but reveals that the occurrence of 1/f, long-range dependence is restricted to only the state of autonomic balance. Both the sympathetic dominant high heart rate state, realized during strenuous experimental exercise, and the parasympathetic dominant low heart rate state, prevalent in (deep) sleep, are characterized by uncorrelated, near white-noise-like scaling, lacking long-range dependence. CONCLUSION: Remarkably, the breakdown of the long-range correlations observed in healthy heart rate in the states of sympathetic and parasympathetic dominance is in stark contrast to the increased correlations which have previously been observed in neurogenic parasympathetic and sympathetic dominance in patients suffering from primary autonomic failure and congestive heart failure, respectively. Our findings further reveal the diagnostic capabilities of heart rate dynamics, by differentiating physiological healthy states from pathology.

Repeated haemorrhages in peripheral nerve sheath tumours of the salivary glands after minor injury.

Repeated haemorrhages in peripheral nerve sheath tumours of the salivary glands are rare. We report the case of a patient with neurofibromatosis type 1 who had two episodes of massive haemorrhage in his right parotid gland the day after a minor injury. Oral and maxillofacial surgeons should be aware that vasculopathy may occur in patients with these tumours.

Fatty acids selectively inhibit eukaryotic DNA polymerase activities in vitro.

The in vitro relationship between eukaryotic DNA polymerases and fatty acids was investigated. Some fatty acids strongly inhibited the activities of DNA polymerase alpha and/or beta in vitro. The kinetics of inhibition by linoleic acid showed that DNA polymerase alpha was non-competitively inhibited with respect to the DNA template and substrate (dTTP), while DNA polymerase beta was inhibited competitively with both DNA and substrate.

Effect of food on the bioavailability of cyclandelate from commercial capsules.

The bioavailability of five capsules of cyclandelate that are commercially available in Japan was determined in ten healthy volunteers by measuring mandelic acid (a main metabolite of cyclandelate) excreted in the urine. Bioinequivalence among the five capsules was demonstrated. The relative cumulative excretion of mandelic acid of the most poorly bioavailable capsule was 38% of the most highly bioavailable capsule. The effect of food on the bioavailability of these two capsules was investigated by use of two different kinds of food, one containing fat and one containing high carbohydrates but very low fat. The bioavailability of the two capsules was increased when subjects consumed both types of food before drug administration, although there was a greater effect on bioavailability with food containing fat. This suggests that the absorption of cyclandelate was incomplete in fasting subjects, even from the capsule with the highest bioavailability. Bioinequivalence between the two capsules remained after postprandial drug administration.

Molecular cloning and expression during development of the Drosophila gene for the catalytic subunit of DNA polymerase epsilon.

We have cloned the genomic DNA and cDNA of Drosophila DNA polymerase epsilon (pol-epsilon) catalytic subunit (GenBank No. AB035512). The gene is separated into four exons by three short introns, and the open reading frame consists of 6660 base pairs (bp) capable of encoding a polypeptide of 2220 amino acid residues. The calculated molecular mass is 255018, similar to that of mammalian and yeast homologues. The deduced amino acid sequence of the pol-epsilon catalytic subunit shares approximately 41% identity with human and mouse homologues as well as significant homology those of C. elegans, S. cerevisiae and S. pombe. Similar to the pol-epsilon catalytic subunits from other species, the pol-epsilon catalytic subunit contains domains for DNA polymerization and 3'-5' exonuclease in the N-terminal region, and two potential zinc-finger domains in the C-terminal regions. Interestingly, a 38 amino acid sequence in the C-terminal region from amino acid positions 1823 to 1861 is similar to the site for Mycoplasma ATP binding and/or ATPase domain (GenBank No. P47365). Northern hybridization analysis indicated that the gene is expressed at the highest levels in unfertilized eggs, followed by zero to 4h embryos and adult females, and then embryos at other embryonic stages, instar larva stages and adult males. Low levels of the mRNA were also detected at the pupa stage. This pattern of expression is similar to those of DNA replication-related enzymes such as DNA polymerase alpha and delta except for the high level of expression in adult males.

Stimulation of the hypothalamic-pituitary-adrenal axis by epidermal growth factor.

The effects of mouse epidermal growth factor (mEGF) on the hypothalamic-pituitary-adrenocortical axis were studied in vivo in conscious male rats and in vitro with cultured anterior pituitary cells. Both intravenous (i.v.) and intracerebroventricular (i.c.v.) injections of mEGF (5-20 ng: 8.3-33.3 pmol) produced significant, dose-related increases in plasma ACTH and corticosterone concentrations. The potency of mEGF is 1/20-1/50 of that of rat corticotropin-releasing factor (rCRF), and pretreatment with 150 micrograms alpha-helical CRF (9-41) completely abolished the effects of the two peptides. mEGF in concentrations ranging from 10 pM to 10 nM did not significantly affect ACTH release from dispersed anterior pituitary cells. It also failed to alter ACTH secretion in response to rCRF. These results indicate that mEGF stimulates the pituitary-adrenocortical axis through a CRF-dependent mechanism.

Intensification of chemotherapy using block therapies as consolidation and reinduction therapies for acute lymphoblastic leukemia during childhood.

Between April 1994 and March 1997, 143 children (age range, 1-15 years) with newly diagnosed acute lymphoblastic leukemia (ALL), except for those patients with t(9;22), were treated according to protocol-94 of the Osaka Childhood Leukemia Study Group. In this trial, the intensity of chemotherapy was enforced in the consolidation and reinduction phases by introducing AML-type block therapies consisting of concentrated administration of 4 to 6 drugs during 5 or 6 days. For patients in the higher risk groups, rotational combination chemotherapy was introduced following the early phase. A total of 124 children with B-cell precursor ALL (B-pre ALL) were classified into 3 groups, the ultrahigh-risk group (UHRG) (15 patients), the high-risk group (HRG) (61 patients), or the standard-risk group (SRG) (48 patients), based on age. leukocyte count, immunophenotype, central nervous system leukemia, response to treatment, and selected chromosomal abnormalities. The complete remission rate was 93%, and the 6-year event-free survival (EFS) rate was 79%+/-4%. EFS rates for the UHRG, HRG, and SRG groups were 67%+/-12%, 80%+/-6%, and 81%+/-6%, respectively. Nineteen patients with T-cell ALL were treated with the protocol for the UHRG. Thirteen patients (68%) attained complete remission, and the 6-year EFS rate was 55%+/-12%. Thus, intensification of chemotherapy improved the EFS rate and AML-type block therapies appeared to be effective as the consolidation and reinduction therapies for B-pre ALL.

Myelodysplastic syndrome after regression of transient abnormal myelopoiesis in a Down syndrome infant: different clonal origin?

A boy with Down syndrome who developed myelodysplastic syndrome after regression of transient abnormal myelopoiesis (TAM) is described. His blast cells in TAM had other chromosome abnormalities in addition to trisomy 21;50,XY,+21c,+12,+14,+21. Serial chromosome analysis in follow-up showed abnormal clones involving monosomy 7. Myelodysplastic syndrome was diagnosed. Because two clones had different karyotypes, they might have derived from different clones.

Effects of neonatal treatment with monosodium glutamate on circadian locomotor rhythm in the rat.

In order to study the effects of treatment with monosodium glutamate (MSG) during the neonatal period on the intrinsic circadian timekeeping system in rats, the locomotor activity of blinded MSG-treated and control (saline-treated) rats was analyzed with power spectral analysis and cross-correlation. In contrast to a robust free-running circadian rhythm in the control rats, a significant shortening of the circadian period and rapid decomposition into ultradian components were noted in the MSG-treated rats. Computer-assisted stereometry of the hypothalamic nuclei revealed that, in addition to the well-known severe damage in the arcuate nuclei (ARC), the volumes of the suprachiasmatic nuclei (SCN) and ventromedial hypothalamic nuclei (VMH) were also reduced significantly in the MSG-treated rats. Although no gross histological damage was apparent in either the SCN and VMH, neonatal MSG treatment appears to impair the function of SCN to integrate many minor oscillations in the brain into a single, definite and precise circadian period.

Endothelin-3 stimulates the hypothalamic-pituitary-adrenal axis.

Endothelin-3 (ET-3) is a member of the novel vasoconstrictive peptide family, identified in porcine central nervous system. Intravenous bolus injection of 1000 pmol/kg of ET-3 in freely moving rats caused significant increases in plasma ACTH and corticosterone levels, almost equivalent to those of 100 pmol/kg of rat corticotropin-releasing hormone (rCRH). The action of ET-3 was virtually abolished by pretreatment of CRH-antagonist, alpha-helical CRH. When ET-3 was added to cultured anterior pituitary cells, neither direct stimulation of ACTH release nor potentiation of rCRH action was noted. The results indicate that ET-3 may function as a neuropeptide and stimulation of the CRH-neurons, direct or inderect, is mainly responsible for activation of ACTH and corticosterone release.

Application of the NONMEM method to evaluation of the bioavailability of drug products.

The NONMEM method, one of the methods used for analysis of population pharmacokinetics, was applied to the evaluation of the relative bioavailability of drug products. The data of 2 x 2 crossover studies of several drugs and a 3 x 3 crossover study of phenytoin using healthy volunteers were analyzed by the NONMEM method, as well as by the confidence interval procedure, which is a standard approach to the bioequivalence test data using model-independent parameters. Very close confidence intervals for the relative difference in bioavailability were estimated by the NONMEM method and the standard approach, both in cases where products were bioequivalent and where they were not. The NONMEM method could also correctly estimate the relative bioavailability of the products using simulated clinical data obtained by randomly reducing the sampling points of the phenytoin data mentioned above, which cannot be analyzed by the standard approach. Thus, the usefulness of the NONMEM method was confirmed for evaluation of bioavailability using clinical or experimental data.

Power analyses of moment analysis parameter in bioequivalence tests.

Simulated and experimental data were used to evaluate the mean residence time (MRT) as a parameter for estimating the rate of bioavailability in bioequivalence tests and to compare MRT with tmax (the time of peak drug concentration), both pharmaceutically and statistically. Although the values of MRT were more dependent on the elimination rate constant than tmax, MRTs were sufficiently sensitive to variations in the absorption rate. The statistical power of MRT infinity obtained by an extrapolation method was lower (especially when the flip-flop phenomenon occurred) than that of MRTt (calculated using data from zero time through the last sampling time). However, the power of MRTt was shown to be comparable to or higher than that of either AUCt or Cmax (the peak drug concentration) from simulated data. These results were also confirmed experimentally using previously obtained data. The effect of variances of plasma concentrations on the power of these parameters was also studied using a simulation technique. Even large variances near the last sampling time did not substantially affect the power of MRTt. Thus MRTt can be used as a parameter for estimating the rate of bioavailability from dosage forms in place of tmax in bioequivalence tests.

Gastric emptying of tablets and granules in humans, dogs, pigs, and stomach-emptying-controlled rabbits.

Rates of gastric emptying of nondigestible tablets and granules in humans were compared with those in three animal models: dogs, minipigs, and stomach-emptying-controlled rabbits. The rates of gastric emptying of both dosage forms in dogs tended to be faster than or similar to those in humans, whereas the rates in pigs were slower. In stomach-emptying-controlled rabbits, no tablets were emptied from the stomach because of their large size. The rate of gastric emptying of granules in rabbits was slow and variable. Food delayed gastric emptying in dogs, especially for tablets. In rabbits, the rate of gastric emptying of granules was faster when the granules were given before feeding, in comparison with that after feeding or under fasting conditions. We concluded that the dog is a better animal model for bioavailability studies under fasting conditions than the pig and the rabbit.

Bioavailability of griseofulvin from tablets in humans and the correlation with its dissolution rate.

Dissolution rates of 10 commercial microsize griseofulvin tablets and one ultramicrosize griseofulvin tablet were preliminarily determined in 18 liters of pH 7.2 phosphate buffer and in 900 ml of 40% dimethylformamide as test media. Addition of dimethylformamide affected the dissolution behavior of the formulations. The products, three microsize and one ultramicrosize, were selected for further studies on the bioavailability in humans and dissolution. Significant differences among the formulations were found in serum levels Cmax, and AUC47.5 hr, but not in AUC infinity and tmax. The maximum difference of Cmax was approximately 40%. The ultramicrosize product showed lower Cmax and serum levels at earlier sampling times than two microsize products. The dissolution rates determined under sink and nonsink conditions without pretreatment significantly correlated with the serum level at 1 hr but not with the other in vivo parameters. Only the dissolution rate determined by the sink method with pretreatment with a small quantity of water (1.0 ml) and plastic beads significantly correlated with serum levels at 3 and 5 hr, Cmax, and AUC 47.5 hr.

Bioavailability of griseofulvin from tablets in beagle dogs and correlation with dissolution rate and bioavailability in humans.

The bioavailability of four griseofulvin tablets in beagle dogs, including an ultramicrosize tablet used previously in a human bioavailability study, was investigated on the basis of the plasma 6-demethyl-griseofulvin concentration. The relations with the in vivo findings in humans and the in vitro dissolution rates also were examined. Contrary to the lower bioavailability of the ultramicrosize formulation in humans, it provided the best bioavailability in beagles. The microsize griseofulvin formulations showed similar in vivo results to those in humans. Poor correlation of in vivo parameters between humans and beagles was attributed to the discrepancy of the availability of the ultramicrosize formulation between the two species. The dissolution rates determined by the pretreatment method using plastic beads were correlated more with the in vivo findings than those determined by the other methods. Beagles were a useful animal model for bioavailability studies of certain griseofulvin formulations but not ultramicrosize ones.

A new DNA polymerase species from Drosophila melanogaster: a probable mus308 gene product.

Harris et al. [P.V. Harris, O.M. Mazina, E.A. Leonhardt, R.B. Case, J.B. Boyd, K.C. Burtis, Molecular cloning of Drosophila mus308, a gene involved in DNA cross-link repair with homology to prokaryotic DNA polymerase I genes, Mol. Cell. Biol., 16 (1996) 5764-5771.] reported the molecular cloning of Drosophila mus308 gene, and its nucleotide and protein sequences similar to DNA polymerase I. In the present study, we attempted to find and isolate the gene product by purifying a DNA polymerase fraction not present in mus308 flies. A new DNA polymerase with properties different from those of any known polymerase species was identified and partially purified from the wild-type fly embryos through ten column chromatographies. The enzyme was resistant to aphidicolin, but sensitive to ddTTP and NEM. Human proliferating cell nuclear antigen (PCNA) and Drosophila replication protein A (RP-A) did not affect the polymerase activity. It preferred poly(dA)/oligo(dT) as a template-primer. The molecular mass was about 230 kDa with a broad peak region of 200 to 300 kDa in HiPrep16/30 Sephacryl S-300 gel filtration. These properties a different from those of all reported Drosophila polymerase classes such as alpha, beta, gamma, delta, epsilon and zeta and closely resemble those of the gene product expected from the nucleotide sequence. The new polymerase species appears to have ATPase and 3'-5' exonuclease activities as shown by the chromatographies.