Clinical and pedigree study on familial cases of West syndrome in Japan.

Nationwide survey on familial cases of West syndrome (WS) in first- and second-degree relatives was conducted by mailing a questionnaire to 64 major university hospitals, children's hospitals, and epilepsy centers in Japan, and by review of the Japanese cases in the literatures. Thirty-four familial cases, 20 males and 14 females, were obtained in 15 families including one with five affected members in two generations and another with three affected male siblings including a half brother by a different father (X-linked WS). A mother and the child or children were involved in three families. Nine families had 21 cryptogenic cases and six families had 13 symptomatic cases, and the etiologies were same among the affected members in each family. Familial cases of WS have characteristic clinical features and genetic mechanisms. Age of onset, seizure types, electroencephalographic abnormalities, early seizure outcome, effective treatment, long-term seizure prognosis, and long-term developmental prognosis were concordant among the affected members in each family. Long-term seizure and developmental prognoses were far better than those in WS in general, with seizure-free rate of 82% and normal mental development rate of 44%. Poor prognosis was limited to specific symptomatic cases. Adrenocorticotropic hormone (ACTH) was a treatment of choice, and even in relapse of WS after ACTH therapy, the patients well responded to antiepileptic drugs. Specific inheritance pattern was difficult to imagine in the majority of the present cases, except for one family with X-linked WS and another family with five patients of maternal inheritance. These results are helpful for the treatment choice and prognostication of clinical course for familial cases of WS.

A novel emulsifier, labrasol, enhances gastrointestinal absorption of gentamicin.

Gentamicin (GM) is an important aminoglycoside antibiotic for the treatment of infections caused by a wide spectrum of aerobic gram-negative bacilli and gram-positive cocci. As a class, the aminoglycosides are poorly absorbed from the gastrointestinal (GI) tract and are commonly used as injectable and topical preparations. This study was aimed at finding the effect of a novel emulsifier, Labrasol, on the absorption of GM from the GI tract of rats. GM formulations were prepared, either as saline solution or as Labrasol microemulsions, and were administrated to rat small intestine and colon. Plasma GM levels following intestinal application were compared to those obtained with intravenous (i.v.) administration. A 5 mg/kg dose of GM preparation containing Labrasol, 1 ml/kg, administrated into colon resulted in the mean AUC of 21.179+/-1.374 microg x h/ml, compared to 7.813+/-0.105 microg x h /ml obtained with i.v. administration of GM, 1 mg/kg. The absolute bioavailability (BA) of the Labrasol preparation was 54.2%. Labrasol facilitates the transmucosal delivery of GM from rat colon by forming microemulsions, and the BA obtained with Labrasol microemulsion was higher than with other surfactants (8.4% for Tween 80 and 3.4% for Transcutol P). Additionally, in vitro permeation studies demonstrated that Labrasol also inhibited the intestinal secretory transport. The effect of Labrasol is ascribed to both (1) enhanced GM absorption from the GI lumen into the systemic circulation and (2) inhibition of efflux of GM from the enterocytes to the GI lumen.

Involvement of NADH/NADPH oxidase in human platelet ROS production.

Platelets play an important role in atherosclerotic and thromboembolic vascular diseases. It has been reported that reactive oxygen species (ROS) could modify platelet function, and platelets themselves have the ability to produce ROS. However, the enzymatic sources of ROS in platelets have not been fully determined. The NADH/NADPH oxidase system was originally identified as the major source of ROS in phagocytes. Recently, it has become evident that this oxidase is functionally expressed not only in phagocytes but also in various cell types. The present study was undertaken to test the hypothesis that NADH/NADPH oxidase might be expressed in human platelets. Lucigenin-enhanced chemiluminescence (L-CL) and electron spin resonance (ESR) method demonstrated that human platelets obtained from healthy volunteers released ROS, and the released ROS were increased by stimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA) or calcium ionophore. Homogenates of human platelets, as well as MEG01 cells, megakaryocytic cell line, had the enzymatic activity to produce superoxide in NADH/NADPH-dependent manners. This enzymatic activity was suppressed by diphenylene iodonium (DPI), an inhibitor of NADH/NADPH oxidase. Western blot analysis demonstrated that platelets and MEG01 cells expressed p22(phox) and p67(phox) proteins, components of NADH/NADPH oxidase. Thus, human platelets have the enzymatic activity of p22(phox)-based NADH/NADPH oxidase, and this oxidase is likely one of the important sources of ROS in platelets.

Lysophosphatidylcholine increases the secretion of matrix metalloproteinase 2 through the activation of NADH/NADPH oxidase in cultured aortic endothelial cells.

Matrix metalloproteinases (MMPs) play a pivotal role in angiogenesis, atherogenesis, vascular remodeling after vascular injury, and instability of atherosclerotic plaque. The present study was undertaken to investigate the effect of lysophosphatidylcholine, a major component of oxidized low density lipoprotein (LDL), on the regulation of MMPs in cultured bovine aortic endothelial cells (BAECs). Furthermore, we explored the potential role of oxidative stress in the regulation of MMP. LPC increased the secretion of gelatinolytic activity, as well as, protein of MMP-2 from BAECs. The stimulation of BAEC with superoxide increased the production of MMP-2 and it also induced its activation. Electron spin resonance (ESR) with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as spin trap agent demonstrated that lysophosphatidycholine (LPC) induced generation of reactive oxygen (ROS) species from BAECs. The inhibition of NADH/NADPH oxidase, one of the potential sources of superoxide in endothelial cells, attenuated the effect of LPC. Our findings suggest that LPC might activate the endothelial NADH/NADPH oxidase to enhance superoxide production, and it might, in turn, enhance MMP-2 induction.

Lysophosphatidylcholine enhances superoxide anions production via endothelial NADH/NADPH oxidase.

Reactive oxygen species (ROS) including superoxide anions (O2(-)) play a key role in atherogenesis, and endothelial cells have the ability to generate ROS. To investigate the enzymatic sources of ROS and the effects of lysophosphatidylcholine (LPC), an atherogenic lipid, we measured ROS production in cultured bovine aortic endothelial cells (BAECs) by the lucigenin-enhanced chemiluminescence (CL) method and electron spin resonance (ESR). BAEC homogenates had the enzymatic activity of NADH/NADPH oxidase. BAECs cultured on microcarrier beads generated O2(-) under basal conditions. The inhibition of NADH/ NADPH oxidase by diphenylene iodonium (DPI) significantly attenuated O2(-) production, whereas no inhibitors of other oxidases suppressed it. Although LPC enhanced O2(-) production approximately 3.1-fold, its action was suppressed by DPI. Tyrosine kinase inhibitors significantly attenuated LPC-induced O2(-) production. ESR with DMPO demonstrated that LPC increased the formation of the DMPO-hydroxyl adduct in dose- and time-dependent manners. These data suggest that the basal production of O2(-) in endothelial cells is mainly mediated by the NADH/NADPH oxidase system and that LPC activates this oxidase to enhance O2(-) production through a tyrosine kinase-dependent pathway. The enhancement of ROS production by LPC is probably involved in its atherogenic property.

A new insulin-mimetic vanadyl complex, (N-pyridylmethylaspartate)oxovanadium(IV) with VO(N2O2) coordination mode, and evaluation of its effect on uptake of D-glucose by Ehrlich ascites tumour cells.

Because it has been confirmed that the vanadyl(IV) ion and its complexes act as insulin mimetics, a new organic vanadyl complex, (N-pyridylmethylaspartate)oxovanadium (VOPASP) with VO(N2O2) coordination mode, was prepared. Development of a simple and rapid in-vitro assay is needed for recognition of potent insulin-mimetic complexes. Treatment of Ehrlich ascites tumour cells with 2-deoxyglucose in the presence of vanadyl sulphate, or other vanadyl complexes with the same coordination mode (VOPASP, bis(picolinate)oxovanadium (VOPA) and bis(6-methyl picolinate)oxovanadium (VOMPA)), in the presence of 2-deoxy-D-[1-3H]glucose ([3H]deoxyglucose), resulted in concentration-dependent uptake of 2-deoxyglucose by the cells. The responses of the cells to the vanadyl complexes were reflected, in part, by results obtained from the free fatty acid-releasing assay using rat adipocytes. These results show that the in-vitro assay with Ehrlich ascites tumour cells provides an accurate and rapid assessment of glucose uptake by the cells. The assay is proposed as a means of predicting the insulin-mimetic activity of the vanadyl complexes and for studying the mechanism of action of the complexes.

Protective effects of baicalein against cell damage by reactive oxygen species.

Baicalein (5,6,7-trihydroxy-2-phenyl-4H-1-benzopyran-4-one), a naturally occurring flavonoid, was found to prevent human dermal fibroblast cell damage induced by reactive oxygen species such as hydrogen peroxide (H2O2), tert-butyl hydroperoxide (BuOOH) and superoxide anions (.O2-) in a concentration-dependent manner, and was more effective than the iron chelator, deferoxamine, hydroxyl radical (.OH) scavengers such as dimethyl sulfoxide (DMSO) and ethanol (EtOH), the lipid peroxidation chain blocker, alpha-tocopherol (Vit. E) and the xanthine oxidase inhibitor, allopurinol. To probe the mechanism of cell defense, the reaction of baicalein with oxygen free radicals was investigated using electron spin resonance (ESR) spectrometry. Baicalein decreased the signal intensities due to the 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) spin adducts of .OH, .O2- and tert-butyl peroxyl (BuOO.) radicals in a concentration-dependent manner. The IC50 values, which are the 50% inhibition concentrations of baicalein for the free radicals, were 10, 45 and 310 microM, respectively. These results suggested that baicalein possesses free radical scavenging ability which prevents the fibroblast damage induced by these free radical species.

Binding affinity of Cu(II)-VP-16 (etoposide) complex and its analogues to DNA and hydroxyl radical generation during DNA strand breaks.

Conformational effects and affinities of VP-16 (etoposide) and its derivatives to DNA in the presence of Cu(II) ion were examined by circular dichroic (CD) spectra. The Cu(II)/Cu(I) redox kinetics and the hydroxyl radical (.OH) generation from the Cu(II)-complexes were estimated by the stopped-flow kinetics. Based on the results, DNA-cleaving activity of Cu(II)-complexes of VP-16 has been shown to be related with binding affinity of the complex to DNA, Cu(II)/Cu(I) redox and .OH generation, emphasising the mechanism of generated .OH attack to DNA.

High-performance liquid chromatographic analysis of aminoglycoside antibiotics.

A simple precolumn derivatisation method for the determination of aminoglycoside antibiotics (AGs) is described. The stability of the o-phthalaldehyde (OPA) derivatives of the AGs obtain using beta-mercaptopropionic acid (beta-MP) was investigated by reversed-phase HPLC. One of the fluorescent derivatives of sisomicin was stable at least for 6 h in 50% methanol under the optimal conditions used (OPA concentration, pH and temperature). When plasma samples spiked with sisomicin were analysed the response was linear in the calibration range of 136-900 micrograms of sisomicin per injected volume (40 microliters). As little as 0.06 micrograms of sisomicin per 1 ml of plasma could be detected with a signal-to-noise ratio > or = 2. The method was also applied to whole blood samples from rabbit after a subcutaneous injection of 1 mg/kg of the AGs using dried blood spots (DBS) on the filter-paper punched discs. The detection of sisomicin and netilmicin in the DBSs on punched discs (10.1 micrograms of whole blood) were 0.053 and 0.50 micrograms per ml of whole blood, respectively (signal-to-noise ratio > or = 2). The method permits a simple collection of blood at the microlitre level and should prove particularly useful for monitoring the AGs in blood at therapeutic levels in geriatric and paediatric patients and could be also used for the preclinical study of the AGs blood levels of a number of mice or rats without killing. An RP-HPLC method using an on-line clean-up procedure for large sample-volume analysis of serum AGs is also described.

Effects of X-ray irradiation on genomic DNA methylation levels in mouse tissues.

Effects of ionizing radiation on the level of genomic DNA methylation in liver, brain and spleen of mouse as well as in two kinds of cultured cells were examined by high-performance liquid chromatography. Ten Gy of whole body X-radiation reduced the 5-methyldeoxycytidine contents by about 40% within 8 hours after irradiation in liver. Similar effects were observed at 4 or 7 Gy of X-ray irradiation. However, no such change was detected in brain, spleen and cultured cells. The data indicate that radiation-induced alteration in genomic DNA methylation is not ubiquitous among different tissues and cells.

Breaks in double-strand DNA by Cu(II) complexes of etoposide (VP-16) and its derivatives, as evaluated by S1 nuclease treatment.

Single-strand breaks (ssb) in double-strand (ds) DNA produced by hydroxyl radicals (.OH) generated by Cu(II) complexes of podophyllotoxin (PD)-related compounds were evaluated using S1 nuclease digestion. Cu(II) complexes of VP-16 (etoposide, 4'-demethylepipodophyllotoxin-9-(4,6-O-ethylidene-beta-D-glucopyra noside)), 4'-demethylepi-PD (DEPD), and syringic acid (SA) exhibited both ssb and ds breaks (dsb) in ColE1-HaeII and pBR322-BglI DNA fragments, in which the number of ssb was found to be more than three times and four times greater than that of dsb, respectively. Cytosine (C)-methylation of cytosine-guanine doublet (CpG) in pBR322-BglI DNA inhibited both ssb and dsb within DNA segments by .OH generated by the Cu(II) complexes.

Alteration of c-fos gene methylation in human gliomas.

In an attempt to find a common DNA alteration occurring in human glioma, we examined DNA methylation in 34 gliomas of various pathological grades and compared them with those in normal cerebral subcortex DNA. The total methylated cytosine levels in the genome did not differ appreciably between the tumors and the normal tissues; however, the degree of DNA methylation in several proto-oncogenes and suppressor oncogenes showed some alterations. Among them, the c-fos gene demonstrated deviation from that of normal tissues in all cases examined, suggesting that the alteration of c-fos gene methylation plays a role in the early steps of human glioma development.

A case of Kabuki make-up syndrome with central diabetes insipidus and growth hormone neurosecretory dysfunction.

A case of a 6 year old boy with Kabuki make-up syndrome with central diabetes insipidus and growth hormone neurosecretory dysfunction is reported. Magnetic resonance imaging revealed abnormal findings of the pituitary gland and stalk. Good catch-up growth was obtained by treatment with growth hormone. These findings suggest that hypothalamic-pituitary dysfunction might be involved in Kabuki make-up syndrome.

Site-specific DNA cleavage by Cu(II) complexes of podophyllotoxin derivatives.

Site-specific DNA cleavage in the presence of Cu(II) complexes of podophyllotoxin derivatives was investigated with a modified Sanger sequencing method. Cu(II) complexes of 4'-demethylepipodophyllotoxin (DEPD) and syringic acid (SA) cleaved M13mp18 single-strand DNA site-specifically at both cytosine (C) and guanine (G) positions in the GC rich regions and C position, respectively, at pH 7.8. The apparent binding constants of calf thymus DNA-Cu(II) complexes estimated by the differential UV-absorption spectra revealed that both Cu(II)-VP-16 and -DEPD complexes bind to DNA more strongly than does the Cu(II)-SA complex.

High-performance liquid chromatographic analysis of methylation changes of CCGG sequence in brain and liver DNA of mice during pre- and postnatal development.

The change of the methylation of CpG in the CCGG sequence of brain and liver DNAs of mice during late fetal and suckling periods was determined by high-performance liquid chromatography using a reversed-phase column and 0.1 M phosphate buffer (pH 6.0) as the mobile phase. The tissue DNA was digested with the restriction enzyme, MspI, and was labeled at the 5'-end with [gamma-32P]ATP. The cpm% of deoxycytidine 5'-monophosphate (5mdCMP) in total CpG dinucleotides was calculated from the equation 5mdCMP/total CCGG (cpm%) = (5mdCMP)MspI,cpm/[(5mdCMP)MspI,cpm + (dCMP)MspI,cpm] x 100. The brain DNA exhibited a significant decrease in CpG methylation at prenatal day 18 but little change after birth. This marked decline of 5mdCMP in the CCGG sequence may be associated with the increase of enzymes before birth. The liver DNA showed considerable change during the late prenatal period. The observed changes of CpG methylation in liver DNA are indicative of the corresponding alterations of enzymes, multinucleate cells and hepatocytes. The results obtained indicate that both brain and liver cells have the development-associated changes in the conformation and transition of DNA around the time of birth.

Effects of energy restriction on age-associated changes of DNA methylation in mouse liver.

DNA methylation is known to change with age in several mammalian species. Here we have examined the effect of dietary energy restriction on this age-associated change in liver DNA of C3H/SHN mice. The total 5-methyldeoxycytidine level in the genome decreased slightly soon after energy restriction started. The effect, however, diminished with time and no appreciable difference was detected at middle and old ages. The degree of methylation at the c-myc gene, on the other hand, was not affected by energy restriction at early periods, but the age-dependent alterations at later ages were repressed. This is a new finding to show that DNA methylation is one of the molecular indices of aging affected by energy restriction. It suggests an importance of DNA methylation in the aging process.

Biological significance of DNA methylation in the ageing process.

In order to understand the possible importance of DNA methylation in ageing, characteristics of its age-associated changes were examined in mouse and man. The total methylated deoxycytidine level in the genome decreased in the senescent period in mouse liver, but not in mouse brain and human liver. The examination of DNA methylation in each individual gene revealed that only a few genes showed alteration in the senescent phase while many genes change in the maturation period. The alterations were gene- and tissue-specific. Comparison of short-living mouse and long-living man for the age-associated changes of the c-myc gene methylation revealed that the rate of change in mouse was about 20 times faster than that in man. This suggests a deep involvement of DNA methylation in ageing. Further investigations into the causes and consequences of the changes would clarify a basic mechanism of ageing.

Changes of DNA methylation in protooncogenes in the process of radiation-induced transformation of mouse m5S/1M cells in vitro.

To assess the importance of changes in DNA methylation in an X-ray-induced cellular transformation process, methylation patterns of five nuclear protooncogenes in fifteen transformant clones were studied and compared to that of the parental non-transformed cell line m5S/1M. All transformants examined revealed an alteration in DNA methylation in some of the genes, although these changes were variable among them. A comparison of cellular characteristics with corresponding DNA methylation changes in different clones suggested that the loss of contact inhibition and the gain of anchorage independency were associated with increases of methylation in many genes, whereas the acquisition of tumorigenicity was often accompanied by a decrease of methylation in the N-myc and c-myc genes. Resultant data indicate that the alteration of DNA methylation is closely related to transformation process, yet how this involvement occurs is complex and remains unclear.

[Neurophysiologic and histopathologic studies in a case of congenital sensory neuropathy with anhidrosis].

A three year-old boy with congenital sensory neuropathy with anhidrosis (CSNA) was described. Sural nerve biopsy specimens revealed an almost complete absence of unmyelinated fibers and a marked decrease of the density of small myelinated fibers with preservation of the density of large myelinated fibers. No evidence of active degeneration of unmyelinated or myelinated fibers was found. Skin biopsy specimens revealed the absence of nerve terminals and fibers innervating sweat glands, although sweat glands seemed to be apparently normal in their morphological findings. Therefore, it was concluded that the absence of pain and temperature sensations with preservation of touch sensation in our patient was compatible with the morphometric findings of nerve fibers of the sural nerve described. Similarly anhidrosis was concluded to be well explained by the absence of the innervation of sweat glands and the vessels around them. On the other hand, electrophysiologic studies, such as motor and sensory nerve conduction, short latency somatosensory evoked potential and auditory brainstem response, in which the function of the large myelinated fibers is presumably tested, were all normal. Therefore, the structure and function of such large myelinated fibers were spared in this case. From clinical viewpoints, electrophysiologic studies described above are useful to differentiate CSNA from other types of congenital sensory neuropathies, in which large myelinated fibers are affected.

Unusual accumulation of copper related to induction of metallothionein in the liver of LEC rats.

Copper (Cu), iron (Fe), zinc (Zn) and manganese (Mn) levels in organs of LEC rats (Long-Evans rats with a cinnamon-like coat color), which develop spontaneous jaundice with hereditary hepatitis, were determined by instrumental neutron activation analysis method. Unusual accumulations of Cu in the liver of LEC rats were found, depending on the age of the animals, the metal concentration being more than approximately 20-40 times those of normal LEA rats (Long-Evans rats with an agouti coat color). Fe and Zn were also accumulated, in addition to Cu, significantly in the LEC rats. The unusual Cu accumulations in the liver of LEC rats were associated with the induction of metallothionein, estimated by radioimmunoassay method, in the liver of LEC rats, rather than that of superoxide dismutase, estimated by electron spin resonance -spin trapping method. These findings suggest that the unusual Cu accumulation in LEC rats is involved in the development of jaundice, hepatic injury and hepatocellular carcinoma.