A 0.18-µm CMOS high-data-rate true random bit generator through ΔΣ modulation of chaotic jerk circuit signals.

A full-custom design of chaos-based True Random-Bit Generator (TRBG) implemented on a 0.18-µm CMOS technology is presented with unique composition of three major components, i.e., (i) chaotic jerk oscillator, (ii) ΔΣ modulator, and (iii) simple pre/post-processing. A chaotic jerk oscillator is a deterministic source of randomness that potentially offers robust and highly random chaotic signals and exhibits a distinctive property of smoothly balanced-to-unbalanced alternation of double-scroll attractors. The continuous-time 2nd-order ΔΣ modulator is introduced as a mixed-signal interface in order to increase a resolution of random bit sequences while no extra clock is required. The ΔΣ modulator is constructed mainly by a folded-cascode amplifier with sufficient gain and phase margin of 64 dB and 83°, respectively, and a high-speed comparator with a time constant of 2.7 ns. An uncomplicated structure of shift-registers is realized as a post-processing process. The bit sequence of the proposed TRBG successfully passes all statistical tests of NIST SP800-22 test suite, and the ultimate output bit rate is 50 Mbps. The physical layout of a chip area is 212.8 × 177.11 µm and the DC power dissipation is 1.32mW using a 1.8-V single supply voltage. This paper therefore offers a potential alternative to a fully embedded cryptographic module in ASIC applications.

Expression level of vascular endothelial growth factor-C and -A in cultured human oral squamous cell carcinoma correlates respectively with lymphatic metastasis and angiogenesis when transplanted into nude mouse oral cavity.

Angiogenesis is induced by various angiogenic factors including vascular endothelial growth factors (VEGFs), such as VEGF-A, -B, -C and -D, and is involved in tumor progression and metastasis. In an effort to define the expression pattern of VEGFs in oral squamous cell carcinoma (OSCC) and its correlation with clinicopathological factors, we determined the expression levels of VEGFs in OSCC cell lines (HSC-2, HSC-3, HSC-4 and OSC-19) by quantitative RT-PCR and examined their relationship with regional lymph node (LN) and distant metastasis, intratumoral microvessel density (MVD) in tumor transplanted nude mice. We found that HSC-2 and OSC-19 expressed significantly higher levels of VEGF-A and VEGF-C, and caused frequent regional LN metastasis and higher MVD than did the other cell lines. Since VEGF-C is a lymphangiogenic factor, these results suggest that expression of VEGF-C is a useful predictor for LN metastasis.

Quantitative trait loci determining weight reduction of testes and pituitary by diethylstilbesterol in LEXF and FXLE recombinant inbred strain rats.

Increasing exposure to environmental endocrine disruptor, xeno-estrogen, is a serious hazard to male reproductive activity. To explore possible genetic control in susceptibility to xeno-estrogen, the weight reduction of testes induced by the continuous administration of a synthetic estrogen, diethylstilbesterol, were investigated by quantitative trait analysis in LEXF and FXLE recombinant inbred strain rats, consisting of 21 independent strains, 9 of their substrains, parental F344/Stm and LE/Stm strains, and (F344 x LE)F1. For the weight of testes, one highly significant quantitative trait locus (QTL) and one significant QTL were mapped on chromosomes 7 and 1, respectively. The QTL on chromosome 7 is closely associated with c-myc. Pituitary weight and serum prolactin were also variable among recombinant inbred strains, but no QTL was detected for them in this study.

Genomic alterations in primary cutaneous melanomas detected by metaphase comparative genomic hybridization with laser capture or manual microdissection: 6p gains may predict poor outcome.

To clarify the correlation of genomic alterations with clinical and histological features, we performed metaphase comparative genomic hybridization analysis on 20 primary cutaneous melanomas, which were obtained by laser capture or manual microdissection, and 16 melanoma cell lines. There were no differences in the average number of aberrations between acral melanomas (AM) and non-AM, although gains of 5q and 11q13 were more frequent (P=0.05) and 10q loss was less frequent (P=0.01) in AM than in non-AM. Although tumor thickness is considered a measurable estimate of clinical expression, there were no differences in the average number of aberrations among 4 groups, classified by thickness of the tumor. While the majority of aberrations were equally distributed among the 4 groups, 6p gains were found only in the thickest tumors. Patients with 6p or 1q gains had a lower overall survival rate than those without them (P=0.0002 or P=0.013). While gains of 1q, 2q, 3p, 3q, 7q, 20p, and 20q were more frequent in the cell lines than in the primary tumors (P<0.01), losses of 6q, 9p, 10p, and 10q were equally found in both cell lines and primary tumors. The present study showed that chromosomal aberrations had already occurred in the thinner tumors, and that 6p and 1q gains may be a prognostic factor.

Role of the LXXLL-motif and activation function 2 domain in subcellular localization of Dax-1 (dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1).

Dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1 (Dax-1, NR0B1) is an orphan nuclear receptor that represses transcription by Ad4 binding protein/steroidogenic factor 1 (Ad4BP/SF-1, NR5A1). Observations on human diseases and the phenotypes of mice, in which the corresponding genes have been disrupted, have elucidated essential roles of these two nuclear receptors in differentiation of steroidogenic tissues. However, little is known about how the functions of these factors are regulated. Here we have examined their subcellular localization and have clarified the molecular mechanisms regulating subcellular localization of Dax-1. Prompted by the finding that nuclear localization of Dax-1 correlates with the presence of Ad4BP/SF-1 in the early stages of pituitary development, we have tested the possibility that interaction between the two factors is essential for the nuclear localization of Dax-1. In vitro studies with cultured cells demonstrated that an interaction involving the LXXLL motifs in the N-terminal repeat region of Dax-1 plays a key role in its subcellular localization. In addition, we found that a mutant form of DAX-1 (L466R), from a patient with adrenal hypoplasia congenita, was defective in nuclear localization in spite of having an intact N terminus. Taken together, the results reveal that the subcellular localization of Dax-1 is influenced by the presence of Ad4BP/SF-1, and that two regions of Dax-1 have important roles for this process.

Androgen-dependent hereditary mouse keratoconus: linkage to an MHC region.

PURPOSE: To better understand the pathogenesis of hereditary keratoconus, an inbred line of spontaneous mutant mice with keratoconus-affected corneas (SKC mice) was established and studied with a multidisciplinary approach. METHODS: Using a mutant mouse with corneas having a keratoconical appearance as the progenitor, an inbred line of SKC mouse was established by repeated sibling mating. Morphology, cell growth, apoptosis and protein expression of SKC mouse corneas were examined. Castration of males and androgen treatment for females were conducted to determine any androgen dependency of the phenotype. Linkage analysis was conducted to reveal the responsible or predisposing gene of SKC mouse keratoconus. RESULTS: Corneas of the SKC mouse resemble those of human eyes with keratoconus. Both are conical and show similar corneal changes, including apoptosis of keratocytes and increased expression of c-fos protein. The SKC mouse phenotype was transmitted in an autosomal recessive manner, although it was observed almost exclusively in males. Intriguingly, female mice showed the phenotype when injected with testosterone, whereas male incidence of the phenotype diminished drastically when mice were castrated. Linkage analysis localized a predisposition locus to an MHC region on mouse chromosome 17, which includes a locus for the gene for sex-limited protein (Slp). CONCLUSIONS: SKC mouse keratoconus is a potential model for a subset of human keratoconus, which is a disease entity with heterogeneous pathogeneses. Alternatively, SKC mouse keratoconus could be a model for other human or mouse-specific keratopathies.

Annulus of collagen fibrils in mouse cornea and structural matrix alterations in a murine-specific keratopathy.

PURPOSE: Mouse corneas were investigated to see whether a limbal annulus of corneal collagen exists as in humans. Mice with corneas predisposed to topographical changes (the SKC strain) were also examined, to establish the size and spacing of stromal collagen fibrils and the integrity of the annulus. METHODS: X-ray diffraction was used to measure collagen fibril spacing and diameter in normal (the BALB/c strain; four male, two female) and SKC (six male and six female) corneas and to identify the degree of preferred collagen orientation at 200- microm intervals across two BALB/c and four SKC corneas. RESULTS: The average collagen fibril diameter measured 35.5 nm in 3-month-old BALB/c corneas, and 36.9 nm and 37.0 nm, respectively, in corneas of age-matched male and female SKC mice. In male and female SKC corneas, average collagen interfibrillar Bragg spacing was significantly higher (64.5 and 59.9 nm, respectively) than in corneas of BALB/c mice (49.7 nm). Circumferentially aligned collagen, indicative of a limbal annulus of fibrillar collagen 2.2 mm in diameter, was identified in mouse cornea. On occasion, this was disturbed in the SKC phenotype. CONCLUSIONS: Collagen fibrils are marginally larger in the corneas of SKC mice than in the corneas of BALB/c mice and are considerably more widely spaced. An annulus of fibrillar collagen probably exists near the limbus of the normal mouse cornea that may help promote biomechanical stability and maintain corneal shape. A loss of structural integrity in the annulus of some SKC mice may predispose the corneas to biomechanical instability and shape changes.

Effect of n-3 fatty acids on serum lipid levels and hepatic fatty acid metabolism in BALB/c.KOR-Apoeshl mice deficient in apolipoprotein E expression.

N-3 fatty acids exert a potent serum lipid-lowering effect in rodents mainly by affecting hepatic fatty acid oxidation and synthesis. However, it has been observed that fish oil and docosahexaenoic acid ethyl ester do not lower serum lipid levels in apolipoprotein E (apoE)-knockout (Apoetm1Unc) mice generated by gene targeting. To test the hypothesis that apoE expression is required for n-3 fatty acid-dependent regulation of serum lipid levels and hepatic fatty acid metabolism, we examined the effect of fish oil and n-3 fatty acid ethyl esters on the activity and gene expression of hepatic enzymes involved in fatty acid oxidation and synthesis using an alternative apoE-deficient mouse model with the BALB/c genetic background (BALB/c.KOR-Apoeshl). ApoE-deficient mice were fed diets containing 9.4% palm oil, fish oil, or 5.4% palm oil and 1% EPA plus 3% DHA ethyl esters for 15 days. In contrast to the reported data on apoE-knockout mice, fish oil and n-3 fatty acid ethyl esters greatly decreased serum triacylglycerol, cholesterol, and phospholipid levels in the Apoeshl mice. The decreases were greater with fish oil than with ethyl esters. The alterations by dietary n-3 fatty acids of serum lipid levels were accompanied by parallel changes in the activity and mRNA levels of enzymes involved in hepatic fatty acid oxidation and synthesis. The reason for the discrepancy between the results of the current study and previous studies is unknown. However, our study at least indicates that a lack of apoE expression does not necessarily accompany deficits in the n-3 fatty acid-dependent regulation of serum lipid levels and hepatic fatty acid metabolism.

Expression of apolipoprotein A1 in colonic adenocarcinoma.

In an effort to find proteins overexpressed in metastatic colonic adenocarcinomas, differential proteome analyses were undertaken using primary and metastatic tumors. Two-dimensional-gel profiles showed a number of spots that appeared in metastatic tumor in liver, but not in primary tumor. Amino acid or mass spectrometric analyses of some of these spots revealed tht they were apolipoprotein A1 (Apo A1), a protein that is normally expressed in liver and small intestine. Further RT-PCR analysis verified the expression of Apo A1 transcript in metastatic tumors and, unexpectedly, expression was also detected in the primary tumors, although the expression was weaker than in metastatic tumors. Consistent with this finding, immunohistological studies detected the weak expression of Apo A1 protein in primary colonic adenocarcinoma, in addition to the strong expression in metastatic tumors in liver and lymph nodes. In the primary tumors, expression was stronger in the deep layer than in the superficial layer, while in normal mucosal epithelial cells expression was barely visible. Further immunohistochemical study revealed that Apo A1 is expressed in some colonic adenocarcinomas of patients with no metastasis, although incidence and expression levels were lower than in carcinomas with metastases. These findings are consistent with the notion that expression of Apo A1 is associated with colonic adenocarcinoma progression, and thus Apo A1 is a potential marker of the aggression.

Specific expression of Gsta4 in mouse cochlear melanocytes: a novel role for hearing and melanocyte differentiation.

Mammalian pigment cells produce melanin as the main pigment. Melanocytes, one of the two types of mammalian pigment cells, differentiate from the neural crest and migrate to a variety of organs during development. Melanocytes exist not only in the skin but also in other sites such as the cochlea where they are essential for hearing. Mitf(mi-bw) is one of the known recessive alleles of the mouse microphthalmia-associated transcription factor (Mitf) locus, which is essential for the development of pigment cells. Homozygous Mitf(mi-bw)/Mitf(mi-bw) mice have a completely white coat with black eyes and are deaf due to the lack of melanocytes. By comparing gene expression profiles in cochleae of wild-type and Mitf(mi-bw)/Mitf(mi-bw) mice, we now demonstrate the specific expression of glutathione S-transferase alpha 4 (Gsta4) in the stria vascularis. Gsta4 encodes one of the cytosolic glutathione S-transferases (GSTs) which participate in detoxification processes of many tissues. This gene is specifically expressed in intermediate cells of the stria vascularis, suggesting a novel function for cochlear melanocytes. Moreover, among mammalian pigment cells, expression of Gsta4 was restricted to cochlear melanocytes, suggesting that melanocytes in various tissues differentiate from one another depending on their location.

A deletion in the endothelin-B receptor gene is responsible for the Waardenburg syndrome-like phenotypes of WS4 mice.

The WS4 mouse is an animal model for human Waardenburg syndrome type 4 (WS4), showing pigmentation anomalies, deafness and megacolon, which are caused by defects of neural crest-derived cells. We have previously reported that the gene responsible for the WS4 mouse is an allele of the piebald mutations of the endothelin B receptor gene (Ednrb). In this study, we examined the genomic sequence of the Ednrb gene in WS4 mice and found a 598-bp deletion in the gene. The deleted region contains the entire region of exon 2 and the 5' part of exon 3 and is flanked by inverted repeat sequences which are suggested to trigger the deletion. We concluded that the deletion in the Ednrb gene is the causative mutation for the phenotype of WS4 mice.

A new mouse model for infantile neuroaxonal dystrophy, inad mouse, maps to mouse chromosome 1.

Infantile neuroaxonal dystrophy (INAD) is a rare autosomal recessive hereditary neurodegenerative disease of humans. So far, no responsible gene has been cloned or mapped to any chromosome. For chromosome mapping and positional cloning of the responsible gene, establishment of an animal model would be useful. Here we describe a new mouse model for INAD, named inad mouse. In this mouse, the phenotype is inherited in an autosomal recessive manner, symptoms occur in the infantile period, and the mouse dies before sexual maturity. Axonal dystrophic change appearing as spheroid bodies in central and peripheral nervous system was observed. These features more closely resembled human INAD than did those of the gad mouse, the traditional mouse model for INAD. Linkage analysis linked the inad gene to mouse Chromosome 1, with the highest LOD score (=128.6) at the D1Mit45 marker, and haplotype study localized the inad gene to a 7.5-Mb region between D1Mit84 and D1Mit25. In this linkage area some 60 genes exist: Mutation of one of these 60 genes is likely responsible for the inad mouse phenotype. Our preliminary mutation analysis in 15 genes examining the nucleotide sequence of exons of these genes did not find any sequence difference between inad mouse and C57BL/6 mouse.

Efficient SNP analysis enabled by joint application of the muTGGE and heteroduplex methods.

Gene science-based diagnoses have become an increasingly realistic option as the state of knowledge has improved regarding the genetic basis of disease. To facilitate the creation of this potential diagnostic tool, researchers have made large-scale detection of point mutations a key issue. Here, we propose an inexpensive and convenient method with a high performance level for this purpose: micro temperature gradient gel electrophoresis (muTGGE)-empowered heteroduplex analysis (muTG-HD). First, muTGGE was shown to separate double-stranded DNA containing single nucleotide polymorphism (SNP) with sufficiently high resolution when used in the mode of perpendicular TGGE. Using human c-Ki-ras and rat p53 DNA, point mutations could be unequivocally detected by muTG-HD when parallel TGGE was employed. The mutation type (such as G/C to A/T), the position of the point mutation (centre or not) and the DNA size (around 100 or 200 bp) were examined and found to be detectable. Thus, muTG-HD could detect point mutations efficiently at a much lower cost by having multiple lanes per gel.

Mouse models for four types of Waardenburg syndrome.

Waardenburg syndrome (WS) is an auditory-pigmentary syndrome caused by a deficiency of melanocytes and other neural crest-derived cells. Depending on a variety of symptoms associated with the auditory-pigmentary symptoms, WS is classified into four types: WS type 1 (WS1), WS2, WS3, and WS4. Six genes contributing to this syndrome--PAX3, SOX10, MITF, SLUG, EDN3 and EDNRB--have been cloned so far, all of them necessary for normal development of melanocytes. Mutant mice with coat color anomalies were helpful in identifying these genes, although the phenotypes of these mice did not necessarily perfectly match those of the four types of WS. Here we describe mice with mutations of murine homologs of WS genes and verify their suitability as models for WS with special interest in the cochlear disorder. The mice include splotch (Sp), microphthalmia (mi), Slugh-/-, WS4, JF1, lethal-spotting (ls), and Dominant megacolon (Dom). The influence of genetic background on the phenotypes of mice mutated in homologs of WS genes is also addressed. Finally, possible interactions among the six WS gene products are discussed.

Hereditary keratoconus-like keratopathy in Japanese wild mice mapped to mouse Chromosome 13.

Human keratoconus is a common corneal disease with non-inflammatory corneal ectasia, and a subset of this disease is heritable. In an effort to establish animal models for this disease, we discovered Japanese keratoconus (JKC) mice among Mishima molosinus (MSM) mice, an inbred strain of Japanese wild mice (Mus musculus molossinus). Typical phenotypic corneas of JKC mice are, like human keratoconus, conical in shape, although the corneas were often associated with a red punctum at the tip. In contrast to human keratoconus, histological examination revealed the inflammatory changes such as infiltration of capillaries and hematocytes in JKC mouse corneas. Although JKC mouse corneal change is probably secondary to keratitis and is a mouse-specific keratopathy, its pathogenesis may be relevant to that of human keratoconus. Linkage analysis mapped the responsible gene at the markers D13Mit21, D13Mit252, D13Mit279, and D13Mit39, which are located between 21.9 and 34.0 cm of the mouse Chr 13. Candidate genes in this region include genes for cathepsins, interleukin, and chemotaxin. Further study of JKC mice may shed light on pathogenesis of human keratoconus.

Effects of taurine on serum cholesterol levels and development of atherosclerosis in spontaneously hyperlipidaemic mice.

1. The effects of two naturally occurring substances, namely taurine and catechins, on serum cholesterol levels and on the progression of atherosclerotic lesions were evaluated using spontaneously hyperlipidaemic (SHL) mice as an animal model of atherogenesis. 2. Twelve weeks treatment of SHL mice with taurine (1% in drinking water) significantly elevated serum high-density lipoprotein-cholesterol (HDL-C) levels without affecting levels of low-density lipoprotein- and very low-density lipoprotein-cholesterol. In addition, taurine suppressed the development of atherosclerotic lesions by 29%, as determined by oil red O-stained areas in cross-sections of the aorta. 3. In contrast, 12 weeks treatment with a catechin mixture had no apparent effect on serum cholesterol levels and on the progression of atherosclerosis. 4. Serum levels of thiobarbituric acid-reactive substances, an index of oxidized substances, significantly decreased from 9.6 to 6.7 nmol/mL following taurine treatment. 5. We suggest that retardation of atherosclerosis by taurine in SHL mice may be related to decreases in oxidized substances and increases in serum HDL-C levels.

A mouse model of Waardenburg syndrome type 4 with a new spontaneous mutation of the endothelin-B receptor gene.

Waardenburg syndrome (WS) is a hereditary auditory-pigmentary syndrome with hearing impairment and pigmentation anomaly of the skin and iris. In addition to these major symptoms, WS type 4 is associated with Hirschsprung disease. To date, three genes responsible for WS4 have been cloned: genes for a transcription factor SOX10, endothelin 3 (EDN3), and endothelin B receptor (EDNRB). We here describe a novel mutant mouse with a mutation of the Ednrb gene, and propose the mouse as an animal model of WS4. These mutants are with mixed genetic background of BALB/c and MSM (an inbred strain of Japanese wild mice) and have extensive white spotting. They died between 2 and 7 weeks after birth owing to megacolon: their colon distal to the megacolon lacked Auerbach's plexus cells. Interestingly, these mutants did not respond to sound, and the stria vascularis of their cochlea lacked intermediate cells, i.e., neural crest-derived melanocytes. Since these symptoms resembled those of human WS4 and were transmitted in autosomal recessive hereditary manner, the mutants were named WS4 mice. Breeding analysis revealed that WS4 mice are allelic with piebald-lethal and JF1 mice, which are also mutated in the Ednrb gene. Mutation analysis revealed that their Ednrb lacked 318 nucleotides encoding Ednrb transmembrane domains owing to deletion of exons 2 and 3. Interaction between endothelin 3 and its receptor is required for normal differentiation and development of melanocytes and Auerbach's plexus cells. We concluded that a missing interaction here led to a lack of these cells, which caused pigmentation anomaly, deafness, and megacolon in WS4 mice.

Identification of a distal enhancer for the melanocyte-specific promoter of the MITF gene.

Waardenburg syndrome (WS) is characterized by deafness and hypopigmentation because of the lack of melanocytes in the inner ear and skin. WS type 2 is associated with mutations in the gene encoding microphthalmia-associated transcription factor (MITF) that is required for melanocyte differentiation. MITF consists of multiple isoforms with different N-termini, one of which is exclusively expressed in melanocytes, named MITF-M. Its N-terminus is encoded by exon 1M that is under the regulation of the melanocyte-specific (M) promoter. Here we identify a distal regulatory region of 298 bp, located 14.5 kb upstream from exon 1M, which enhances the M promoter activity in cultured melanoma cells. This enhancer activity depends on the proximal M promoter region (-120 to -46). The MITF-M distal enhancer (MDE), thus identified, contains the binding sites for SOX10, a transcription factor responsible for another type of WS, known as Waardenburg-Hirschsprung syndrome. Characterization of MDE has suggested SOX10 as one of factors that are involved in the function of MDE. A putative MDE counterpart is located 12 kb upstream from mouse exon 1M and its role is discussed in relevance to the pathogenesis of red-eyed white Mitf mi-rw mice that exhibit small red eyes and white coat. Moreover, by in situ hybridization analysis, we suggest that Sox10 and Mitf-M (mRNA) are expressed in melanoblasts migrating toward the otic vesicle (prospective inner ear) of mouse embryos but are separately expressed in different cell types of the newborn cochlea. Thus, SOX10 regulates transcription from the M promoter in a developmental stage-specific manner.