Mutations in RECQL4 cause a subset of cases of Rothmund-Thomson syndrome.

Rothmund-Thomson syndrome (RTS; also known as poikiloderma congenitale) is a rare, autosomal recessive genetic disorder characterized by abnormalities in skin and skeleton, juvenile cataracts, premature ageing and a predisposition to neoplasia. Cytogenetic studies indicate that cells from affected patients show genomic instability often associated with chromosomal rearrangements causing an acquired somatic mosaicism. The gene(s) responsible for RTS remains unknown. The genes responsible for Werner and Bloom syndromes (WRN and BLM, respectively) have been identified as homologues of Escherichia coli RecQ, which encodes a DNA helicase that unwinds double-stranded DNA into single-stranded DNAs. Other eukaryotic homologues thus far identified are human RECQL, Saccharomyces cerevisiae SGS1 and Schizosaccharomyces pombe rqh1. We recently cloned two new human helicase genes, RECQL4 at 8q24.3 and RECQL5 at 17q25, which encode members of the RecQ helicase family. Here, we report that three RTS patients carried two types of compound heterozygous mutations in RECQL4. The fact that the mutated alleles were inherited from the parents in one affected family and were not found in ethnically matched controls suggests that mutation of RECQL4 at human chromosome 8q24.3 is responsible for at least some cases of RTS.

Fast x-ray detector system with simultaneous measurement of timing and energy for a single photon.

We developed a fast X-ray detector system for nuclear resonant scattering (NRS) experiments. Our system employs silicon avalanche photo-diode (Si-APD) as a fast X-ray sensor. The system is able to acquire both timing and energy of a single X-ray photon simultaneously in a high rate condition, 106 counts per second for one Si-APD. The performance of the system was investigated in SPring-8, a synchrotron radiation facility in Japan. Good time resolution of 120 ps (FWHM) was achieved with a slight tail distribution in the time spectrum by a level of 10-9 at 1 ns apart from the peak. Using this system, we successfully observed the NRS from the 26.27-keV level of mercury-201, which has a half-life of 630(50) ps. We also demonstrated the reduction of background events caused by radioactive decays in a radioactive sample by discriminating photon energy.

Human RecQ5beta, a large isomer of RecQ5 DNA helicase, localizes in the nucleoplasm and interacts with topoisomerases 3alpha and 3beta.

The RecQ helicase superfamily has been implicated in DNA repair and recombination. At least five human RecQ-related genes exist: RecQ1, BLM, WRN, RecQ4 and RecQ5. Mutations in BLM, WRN and RecQ4 are associated with Bloom, Werner and Rothmund-Thomson syndromes, respectively, involving a predisposition to malignancies and a cellular phenotype that includes increased chromosome instability. RecQ5 is small, containing only a core part of the RecQ helicase, but three isomer transcripts code for small RecQ5alpha (corresponding to the original RecQ5 with 410 amino acids), new large RecQ5beta (991 amino acids) and small RecQ5gamma (435 amino acids) proteins that contain the core helicase motifs. By determining the genomic structure, we found that the three isoforms are generated by differential splicing from the RecQ5 gene that contains at least 19 exons. Northern blot analysis using a RecQ5beta-specific probe indicates that RecQ5beta mRNA is expressed strongly in the testis. Immunocytochemical staining of three N-terminally tagged RecQ5 isomers expressed in 293EBNA cells showed that RecQ5beta migrates to the nucleus and exists exclusively in the nucleoplasm, while the small RecQ5alpha and RecQ5gamma proteins stay in the cytoplasm. Immunoprecipitation and an extended cytochemical experiment suggested that the nucleoplasmic RecQ5beta, like yeast Sgs1 DNA helicase, binds to topoisomerases 3alpha and 3beta, but not to topoisomerase 1. These results predict that RecQ5beta may have an important role in DNA metabolism and may also be related to a distinct genetic disease.

Differential regulation of human RecQ family helicases in cell transformation and cell cycle.

Three human RecQ DNA helicases, WRN, BLM and RTS, are involved in the genetic disorders associated with genomic instability and a high incidence of cancer. RecQL1 and RecQL5 also belong to the human RecQ helicase family, but their correlation with genetic disorders, if any, is unknown. We report here that in human B cells transformed by Epstein-Barr virus (EBV), human fibroblasts and umbilical endothelial cells transformed by simian virus 40, the expression of WRN, BLM, RTS and RecQL1 was sharply up-regulated. In B cells this expression was stimulated within 5-40 h by the tumor promoting agent phorbol myristic acetate (PMA). Interestingly, RecQL5beta, an alternative splicing product of RecQL5 with a nuclear localization signal, is expressed in resting B cells without significant modulation of its synthesis by EBV or PMA, suggesting it has a role in resting cells. We also roughly determined the number of copies per cell for the five RecQ helicase in B cells. In addition, levels of the different RecQ helicases are modulated in different ways during the cell cycle of actively proliferating fibroblasts and umbilical endothelial cells. Our results support the view that the levels of WRN, BLM, RTS and RecQL1 are differentially up-regulated to guarantee genomic stability in cells that are transformed or actively proliferating.

Physical map of the human chromosome 8p12-p21 encompassing tumor suppressor and Werner's syndrome gene loci.

Detailed physical maps of the human genome are important resources for identification and isolation of genes responsible for diseases and for the study of their structure and function. We constructed a 2.0-Mb high-resolution physical map within the human chromosome 8p12-p21 region extending from marker D8S131 to D8S283. The map comprises a series of contigs mostly P1/PAC clones, which span the loci of potential tumor suppressor genes and the Werner's syndrome gene. Each P1/PAC DNA was defined by its size, restriction sites, terminal sequences, intermarker distances and location relative to major genes and markers. The genes on these P1/PAC DNAs were analyzed by an exon amplification method to determine their locations. The genes newly found by the exon amplification method together with other known genes, including those of glutathion reductase, a general transcription factor, protein phosphatase 2A beta subunit and Werner's syndrome, were precisely mapped within the contigs. These P1/PAC DNAs are useful reagents for the generation of new microsatellite markers to narrow the candidate region of the tumor suppressor gene(s) and/or genes responsible for other diseases, which are believed to exist in this region by linkage analysis.

Cloning and characterization of a novel gene, WS-3, in human chromosome 8p11-p12.

A novel human gene referred to as the WS-3 gene, in the short arm of human chromosome 8, was cloned by a combination of exon trapping, thermal asymmetric interlaced-PCR (TAIL-PCR) and the Marathon-Ready cDNA amplification method. The gene consists of 7 exons separated by 6 introns, and is at the telomere side of the STS marker, D8S1055. The full-length WS-3 gene contains 1052 nucleotides and codes for a protein of 190 amino acids with a calculated mol. wt. of 20,747. Southern blot experiments showed that the WS-3 gene exists as a single copy in the human genome. A protein encoded by the WS-3 gene has an R-G-D (Arg-Gly-Asp) motif in the N-terminal region, which seems to confer adhesive properties to macromolecular proteins like fibronectin. Although WS-3 is a small gene with unknown biological function, its ubiquitous expression in various tissues and organs suggests that the encoded protein is one of the essential components of all organs and tissues.

Rothmund-Thomson syndrome due to RECQ4 helicase mutations: report and clinical and molecular comparisons with Bloom syndrome and Werner syndrome.

Rothmund-Thomson syndrome (RTS), an autosomal recessive disorder, comprises poikiloderma, growth deficiency, some aspects of premature aging, and a predisposition to malignancy, especially osteogenic sarcomas. Two kindreds with RTS were recently shown to segregate for mutations in the human RECQL4 helicase gene. We report identification of a new RTS kindred in which both brothers developed osteosarcomas. Mutation analysis of the RECQL4 gene was performed on both brothers and both parents. The brothers were shown to be compound heterozygotes for mutations in the RECQL4 gene, including a single basepair deletion in exon 9 resulting in a frameshift and early termination codon and a base substitution in the 3-prime splice site in the intron-exon boundary of exon 8, which would be predicted to cause a deletion of at least part of a consensus helicase domain. Each parent was shown to be a heterozygote carrier for one mutation. This report strengthens the association between mutations in RECQL4 helicase gene and RTS. Two other recessive disorders, Bloom syndrome and Werner syndrome, are known to be due to other human RECQ helicase gene mutations. These three disorders all manifest abnormal growth, premature aging, and predisposition to site-specific malignancies. The clinical and molecular aspects of RTS, Bloom syndrome, and Werner syndrome are compared and contrasted.

Nuclear resonance scattering of synchrotron radiation by 40K

We have observed the excitation of the 29.83 keV nuclear level of the radioactive 40K nuclide, which level is not populated by any radioactive source, by synchrotron radiation for the first time. The absolute energy of the level of 29.834+/-0.011 keV and the half-life of 4.13+/-0.12 ns were obtained from our measurements. Our success in observing the nuclear excitation of radioactive 40K in a KCl powder sample shows the possibility for study of the local vibrational states and the electronic states of potassium in various areas of research.

Observation of nuclear excitation by electron transition in 197Au with synchrotron X rays and an avalanche photodiode

We have succeeded in observing nuclear excitation by electron transition (NEET) in 197Au by a new method. Monochromatic x-rays of synchrotron radiation were used to ionize the K shell of gold atoms in a target foil. The internal-conversion electrons emitted from excited nuclei were detected with a silicon avalanche photodiode. At a photon energy of 80.989 keV, the NEET probability in 197Au was determined to be (5.0+/-0.6)x10(-8) from a comparison of the event number per photon between NEET and the nuclear resonance at 77.351 keV.

Inhibitory specificity against various trypsins and stability of ovomucoid from Japanese quail egg white.

The inhibitory specificity and stability of ovomucoid from Japanese quail egg white (OMJPQ) were examined to understand its nutritional significance. OMJPQ showed strong inhibitory activities toward trypsins from various origins including human, and the trypsin inhibitions occurred at molar ratios of enzyme to inhibitor between 1/1 and 2/1. On the other hand, an equimolar mixture of the second and third domains of OMJPQ inhibited bovine trypsin more strongly than the corresponding native OMJPQ did. This distinction was partly explained by the presence of steric hindrance on the formation of a 2:1 trypsin-OMJPQ complex. OMJPQ retained about 100% of its original activity over a pH range from 1 to 12 after a 24-h incubation at 37 degrees C. The inhibitor was most thermostable between pH 2 and 5, where more than 70% of its original activity was maintained after a 1-h incubation at 100 degrees C and about 25% of the activity even after a 30-min incubation at 121 degrees C. OMJPQ was also considerably resistant to pepsin attack. Pepsin digestion of the protein resulted in only about 40% loss of the original trypsin-inhibitory activity even after a 24-h digestion. Furthermore, the addition of bovine serum albumin to the digestion mixture brought about rapid elevation in the trypsin-inhibitory activity during an initial 30-min digestion. SDS-PAGE and immunoblot suggested that this was due to the liberation of active inhibitory domains from the native molecule by inter-domain proteolysis.

Serum alpha-fetoprotein values in dogs with various hepatic diseases.

Serum alpha-fetoprotein (AFP) values were measured in hepatic diseased dogs with or without tumor and non-hepatic tumor bearing dogs by a sandwich ELISA using anti-dog AFP antiserum. Serum AFP values were less than 70 ng/ml in clinically healthy dogs. The values in dogs with hepatocellular carcinoma were higher than 1,400 ng/ml in 7 of 9 dogs, wherever those in two dogs with cholangiocarcinoma were in the normal range. Serum AFP values in hepatic diseased dogs without tumor were also high, however, the values were below 500 ng/ml in 90% of the dogs. In non-hepatic tumor dogs, serum AFP values were less than 500 ng/ml in 76% of the dogs. In the surgically removal cases with hepatocellular carcinoma, serum AFP values rapidly decreased. These results suggested that the sandwich ELISA using anti-dog AFP antiserum was an available method for diagnosis of hepatocellular carcinoma in dogs.

Role of platelet activating factor in development of thrombocytopenia and neutropenia in dogs with endotoxemia.

OBJECTIVE: To determine the role of platelet activating factor (PAF) in lipopolysaccharide (LPS)-induced thrombocytopenia and neutropenia in dogs. ANIMALS: 42 dogs. PROCEDURES: Blood samples were obtained from dogs given LPS (40 microg/kg of body weight; n = 16), PAF (1 microg/kg; 6), PAF (5 microg/kg/h for 90 minutes; 4), or physiologic saline (0.9% NaCl) solution (0.1 ml/kg/h for 90 minutes; 3) IV to monitor changes in blood cell counts, using automated counters and blood smears stained with Giemsa. Blood samples were also obtained from dogs given LPS (40 microg/kg) that had (n = 5) or had not (6) been treated beforehand with TCV-309, a potent PAF antagonist. Concentration of PAF in blood was determined by use of 125I-radioimmunoassay in dogs given LPS at 1 mg/kg (n = 3) and 40 microg/kg (9). RESULTS: Thrombocytopenia and neutropenia were found in all dogs except those given saline solution. The LPS-induced thrombocytopenia was significantly suppressed by prior treatment with TCV-309. The PAF concentrations increased markedly 1 hour after injection of 1 mg/kg of LPS and increased slightly but significantly 10 minutes after injection of 40 microg/kg of LPS. CONCLUSION AND CLINICAL RELEVANCE: PAF plays an important role in the development of LPS-induced thrombocytopenia and neutropenia in dogs. Control of PAF production, PAF-induced effects, or both may be important in the treatment of dogs with gram-negative bacterial infections and associated thrombocytopenia and neutropenia.

Physiological levels of C-reactive protein in normal canine sera.

This study was undertaken to investigate whether the level of C-reactive protein (CRP) in the serum of dogs undergoes physiological variation, using 10 normal Beagle dogs (5 males and 5 females), 1-2 years old, maintained in a healthy condition in a controlled environment. The CRP concentration in the sera collected seven times each day at intervals of approximately 3 h ranged from 0.8 to 16.4 micrograms/ml (mean 5.06 +/- 3.60) in one experiment and from 0.8 to 14.0 micrograms/ml (mean 4.50 +/- 2.80) in a second experiment. On examining the 24-h variations in the concentration of CRP in serum, neither consistent changes nor a definite pattern of circadian rhythm was detected. During 28 days observation, only very slight changes, which seemed attributable to analytical errors, were seen in any of the dogs, except one. The concentration of CRP in the serum during the 28 days ranged from 0.8 to 22.6 micrograms/ml (mean 3.65 +/- 1.40). The concentrations underwent no significant variations in individual dogs, but significant differences were found between the dogs (p < 0.01).

Immunological determination of faecal haemoglobin concentrations in dogs.

Faecal haemoglobin (Hb) concentrations in apparently healthy experimental Beagle dogs and in dogs of various breeds kept in private households or at breeders were measured by reversed passive latex agglutination (RPLA) and enzyme-linked immunosorbent assay (ELISA) in an effort to define the physiological concentrations of faecal Hb in the dog. In 88% (53) of 60 experimental Beagle dogs (30 males and 30 females), the RPLA titres were 1:2 and 1:8 and the faecal Hb concentrations ranged from 40.0 to 431.5 (mean 184.1 +/- 92.6) micrograms/g faeces by ELISA. No significant difference was found in Hb levels or RPLA titres between males and females. Seven dogs (12%) had significantly greater RPLA titres and Hb concentrations by ELISA than the remaining dogs. In 84% (45) of the 53 dogs kept in private households or at breeders, the RPLA titres were < 1:1 to 1:8 and the faecal Hb concentrations ranged from 7.1 to 456.7 (mean 137.5 +/- 128.7) micrograms/g faeces in ELISA. Eight of these dogs (15.1% of 53 dogs) had significantly greater RPLA titres and Hb concentrations by ELISA than the remaining dogs. There were no significant differences between the Beagles and dogs kept in private households or at breeders. In conclusion, in 98 (86.7% of 113) dogs the physiological concentrations of RPLA titres were < 1:1 to 1:8 and the faecal Hb concentrations were 143.5-185.1 micrograms/g (95% confidence level). Approximately 13.3% of apparently healthy dogs had higher faecal Hb concentrations, suggesting the presence of subclinical haemorrhages. Four dogs suffering from colorectal cancer also had high faecal Hb concentrations.

Evaluation of Parkinson disease and Alzheimer disease with the use of neuromelanin MR imaging and (123)I-metaiodobenzylguanidine scintigraphy.

BACKGROUND AND PURPOSE: Progressive changes in the substantia nigra pars compacta and locus ceruleus of patients with Parkinson disease and Alzheimer disease visualized by neuromelanin MRI and cardiac postganglionic sympathetic nerve function on (123)I-metaiodobenzylguanidine scintigraphy have not been fully evaluated. We compared the diagnostic value of these modalities among patients with early Parkinson disease, late Parkinson disease, and Alzheimer disease. MATERIALS AND METHODS: We compared contrast ratios of signal intensity in medial and lateral regions of the substantia nigra pars compacta and locus ceruleus with those of the tegmentum of the midbrain and the pons, respectively, by use of neuromelanin MRI in patients with early Parkinson disease (n = 13), late Parkinson disease (n = 31), Alzheimer disease (n = 6), and age-matched healthy control subjects (n = 20). We calculated heart-to-mediastinum ratios on (123)I-metaiodobenzylguanidine scintigrams after setting regions of interest on the left cardiac ventricle and upper mediastinum. RESULTS: The signal intensity of the lateral substantia nigra pars compacta on neuromelanin MRI was significantly reduced in early and late Parkinson disease, and that of the medial substantia nigra pars compacta was gradually and stage-dependently reduced in Parkinson disease. The signal intensity of the locus ceruleus was obviously reduced in late Parkinson disease. Signal reduction was not significant in the substantia nigra pars compacta and locus ceruleus of patients with Alzheimer disease. The heart-to-mediastinum ratio on (123)I-metaiodobenzylguanidine scintigrams was stage-dependently reduced in Parkinson disease and normal in Alzheimer disease. The signal intensity ratios in substantia nigra pars compacta and locus ceruleus on neuromelanin MRI positively correlated with the heart-to-mediastinum ratio on (123)I-metaiodobenzylguanidine scintigrams. CONCLUSIONS: Both neuromelanin MRI and (123)I-metaiodobenzylguanidine scintigraphy can help to evaluate disease progression in Parkinson disease and are useful for differentiating Parkinson disease from Alzheimer disease.

α-D-Glucosyl Transfer to Phenolic Compounds by Sucrose Phosphorylase from Leuconostoc mesenteroides and Production of α-Arbutin.

Transglycosylation from sucrose to phenolic and related compounds by sucrose phosphorylase (EC 2.4.1.7) from Leuconostoc mesenteroides was studied. The enzyme had a rather broad acceptor specificity and transferred glucosyl residue of sucrose to various acceptors such as hydroxybenzenes, hydroxybenzoic acids, benzyl alcohol, and hydroxybenzyl alcohols. The enzyme could transfer the glucosyl moiety of sucrose to phenolic OH groups and alcoholic (hydroxymethyl) OH groups, though not to carboxyl groups. The phenolic OH group was more effective than a hydroxymethyl group as the acceptor. Phenolic OH groups adjacent to hydroxyl, hydroxymethyl, or carboxyl groups had an increased effectiveness as acceptors. About 2.3 g of the purified transfer product was obtained from 2.0 g of hydroquinone. Its structure was identified as hydroquinone-O-α-d-glucopyranoside (α-arbutin) on the bases of the secondary ion mass spectrometry analysis, the component analysis of its enzymatic hydrolysates, and the carbon-13 nuclear magnetic resonance analysis. The browning resistance of α-arbutin to light irradiation was extremely increased compared to that of hydroquinone. The inhibitory activity on tyrosinase of α-arbutin was almost equal to that of arbutin.

Discrimination shift learning and response inhibition of moderately and severely mentally retarded and non-retarded children.

Simple two-choice discrimination shift learning performances of 20 moderately and severely mentally retarded children were compared with those of 20 non-retarded ones. A learning task arranged with a pair of yellow circle and green triangle slides was used. No difference was found between mentally retarded and non-retarded children in the learning rate of initial discrimination, but the mentally retarded attained shift discrimination more slowly than the non-retarded. Also, the extinction of initial positive responses in mentally retarded children was slower than in non-retarded children. It was suggested that the difficulty of discrimination shift in severely subnormal children is due to failure to suppress a previously learned response to prior positive cues.

Verbalization of appropriate and inappropriate cues in discrimination reversal learning by moderately mentally retarded children.

Effects of verbalization on discrimination reversal learning for moderately mentally retarded children were examined under three training conditions. Predicted results indicated that verbalization of both appropriate and inappropriate cues within relevant dimension significantly facilitated reversal shift performance, as compared to verbalization of only appropriate cues or non-verbalization. Results were interpreted to be due to the fact that verbalization of both appropriate and inappropriate cues actually served to utilize attentional or verbal mediators in terms of combining appropriate with inappropriate cues within relevant stimulus dimension.

HTLV-1 Tax protein interacts with cyclin-dependent kinase inhibitor p16INK4A and counteracts its inhibitory activity towards CDK4.

Tax, a regulatory protein of human T-cell leukemia virus type 1 (HTLV-1), is an oncoprotein which immortalizes human T cells and induces tumors in transgenic mice. These effects may be due to its interaction with cellular proteins, consisting of several transcription factors including CREB, NF-kappa B and SRF, and the transcriptional inhibitor, I kappa B. Here, we found that Tax binds to a cyclin-dependent kinase inhibitor, p16INK4A, which has ankyrin motifs similar to I kappa B. p16INK4A binds to the cyclin-dependent kinases, CDK4 and CDK6, and inhibits their activity, resulting in suppression of G1 phase progression. The binding of Tax to p16INK4a induced a reduction in the p16INK4A-CDK4 complex, with subsequent activation of CDK4 kinase. Tax also suppressed p16INK4A-mediated inhibition of U2OS cell growth. The p16INK4A gene was frequently deleted in many T-cell lines, but not in HTLV-1-infected T-cell lines. Taking these findings together, the functional inactivation of p16INK4A by Tax through protein-protein interaction is suggested to contribute to cellular immortalization and transformation induced by HTLV-1 infection.