Element Selectivity in Second-Harmonic Generation of GaFeO_{3} by a Soft-X-Ray Free-Electron Laser.

Nonlinear optical frequency conversion has been challenged to move down to the extreme ultraviolet and x-ray region. However, the extremely low signals have allowed researchers to only perform transmission experiments of the gas phase or ultrathin films. Here, we report second harmonic generation (SHG) of the reflected beam of a soft x-ray free-electron laser from a solid, which is enhanced by the resonant effect. The observation revealed that the double resonance condition can be met by absorption edges for transition metal oxides in the soft x-ray range, and this suggests that the resonant SHG technique can be applicable to a wide range of materials. We discuss the possibility of element-selective SHG spectroscopy measurements in the soft x-ray range.

Electroencephalography in the monitoring of lithium toxicity: A case report.

WHAT IS KNOWN AND OBJECTIVE: If lithium toxicity occurs during treatment by lithium, treatment is performed while monitoring serum lithium levels. CASE SUMMARY: We report a case of lithium toxicity in a patient with bipolar affective disorder who developed neurotoxic symptoms associated with elevated serum lithium level and abnormal electroencephalography (EEG) changes. The elevated serum lithium level decreased before EEG normalization, which was associated with disappearance of all neurotoxic symptoms. WHAT IS NEW AND CONCLUSION: Monitoring EEG changes is useful for the diagnosis and treatment of lithium toxicity accompanied by abnormal EEG changes.

Long-acting formulation leading to severe long-term adverse effects: a case report of fluphenazine and persistent extrapyramidal symptoms.

WHAT IS KNOWN AND OBJECTIVE: Long-acting formulations are an important therapeutic option for non-adherent patients with schizophrenia. There is a commonly held view that management of long-acting formulation-induced side effects is difficult. CASE DESCRIPTION: We present a patient with schizophrenia who developed acute and persistent extrapyramidal symptoms requiring tracheostomy and long-term rehabilitation after long-acting injections of fluphenazine decanoate. Extrapyramidal symptoms improved with declining fluphenazine concentration and antiparkinsonian drug therapy with bromocriptine. WHAT IS NEW AND CONCLUSION: Long-acting formulations may lead to severe persistent adverse effects. For preventing fluphenazine-induced side effects, a possible option might be the antiparkinsonian drug therapy with bromocriptine.

The canine prostate cancer cell line CHP-1 shows over-expression of the co-chaperone small glutamine-rich tetratricopeptide repeat-containing protein α.

Although androgen therapy resistance and poor clinical outcomes are seen in most canine prostate cancer cases, there are only a few tools for analysing canine prostate cancer by using a cell biological approach. Therefore, to evaluate androgen-independent neoplastic cell growth, a new canine prostate cancer cell line (CHP-1) was established in this study. CHP-1 over-expressed the co-chaperone small glutamine-rich tetratricopeptide repeat-containing protein α (SGTA), which is over-expressed in human androgen-independent prostate cancer. The CHP-1 xenograft also showed SGTA over-expression. Although CHP-1 shows poor androgen receptor (AR) signalling upon dihydrotestosterone stimulation, forced expression of AR enabled evaluation of AR signalling. Taken together, these results suggest that CHP-1 will be a useful model for investigating the pathogenesis of androgen-dependent and androgen-independent canine prostate cancer.

Properties of the feline tumour suppressor reduced expression in immortalized cells (REIC/Dkk-3).

Reduced expression in immortalized cells (REIC/Dkk-3), a member of the human Dickkopf (Dkk) family, is a growth suppressor in human and canine mammary tumours. Mammary gland tumours are common neoplasms with high malignancy in female cats. The purpose of this study was to clone the feline REIC/Dkk-3 homolog, investigate its expression in cell lines established from feline mammary gland tumours, and test its tumour suppressor function. Western blot analysis revealed that expression of the REIC/Dkk-3 protein was reduced in feline mammary carcinoma cell lines. Forced expression of REIC/Dkk-3 induced apoptosis in feline mammary tumour cell lines. These results demonstrate that REIC/Dkk-3 expression, which is downregulated in feline mammary tumour cell lines, results in the induction of apoptosis in these cells. Our findings suggest that feline REIC/Dkk-3 represents a potential molecular target for the development of therapies against feline mammary cancers.

Fluvoxamine alleviates ER stress via induction of Sigma-1 receptor.

We recently demonstrated that endoplasmic reticulum (ER) stress induces sigma-1 receptor (Sig-1R) expression through the PERK pathway, which is one of the cell's responses to ER stress. In addition, it has been demonstrated that induction of Sig-1R can repress cell death signaling. Fluvoxamine (Flv) is a selective serotonin reuptake inhibitor (SSRI) with a high affinity for Sig-1R. In the present study, we show that treatment of neuroblastoma cells with Flv induces Sig-1R expression by increasing ATF4 translation directly, through its own activation, without involvement of the PERK pathway. The Flv-mediated induction of Sig-1R prevents neuronal cell death resulting from ER stress. Moreover, Flv-induced ER stress resistance reduces the infarct area in mice after focal cerebral ischemia. Thus, Flv, which is used frequently in clinical practice, can alleviate ER stress. This suggests that Flv could be a feasible therapy for cerebral diseases caused by ER stress.

Detection of pulsation in unruptured cerebral aneurysms by ECG-gated 3D-CT angiography (4D-CTA) with 320-row area detector CT (ADCT) and follow-up evaluation results: assessment based on heart rate at the time of scanning.

PURPOSE: Many epidemiological studies on unruptured cerebral aneurysms have reported that the larger the aneurysm, the higher the risk of rupture. However, many ruptured aneurysms are not large. Electrocardiography (ECG)-gated 3D-computed tomography angiography (4D-CTA) was used to detect pulsation in unruptured cerebral aneurysms. The differences in the clinical course of patients in whom pulsation was or was not detected were then evaluated. METHODS: Forty-two patients with 62 unruptured cystiform cerebral aneurysms who underwent 4D-CTA and follow-up 3D-CTA more than 120 days later were studied. The tube voltage, tube current, and rotation speed were 120 kV, 270 mA, and 0.35 s/rot., respectively. ECG-gated reconstruction was performed, with the cardiac cycle divided into 20 phases. Patients with heart rates higher than 80 bpm were excluded, so 37 patients with 56 aneurysms were analyzed. RESULTS: Pulsation was detected in 20 of the 56 unruptured aneurysms. Of these 20 aneurysms, 6 showed a change in shape at the time of follow-up. Of the 36 aneurysms in which pulsation was not detected, 2 showed a change in shape at follow-up. There was no significant difference in the follow-up interval between the two groups. The aneurysms in which pulsation was detected were significantly more likely to show a change in shape (P = 0.04), with a higher odds ratio of 7.286. CONCLUSION: Unruptured aneurysms in which pulsation was detected by 4D-CTA were more likely to show a change in shape at follow-up, suggesting that 4D-CTA may be useful for identifying aneurysms with a higher risk of rupture.

Efficacy of a 5-HT1a receptor agonist in atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) can be aggravated by mental stress. We recently showed that pretreatment with tandospirone citrate (TC), a serotonin (5-HT) agonist for the 5-HT(1A) receptor subtype, significantly inhibits stress-induced degranulation of mouse dermal mast cells. AIMS: To evaluate the efficacy of TC in treatment of AD. METHODS: Changes in anxiety levels, depression symptoms and the clinical severity of AD after administration of TC were examined. Data were collected for 20 patients with AD who received TC 30 mg/day for 4 weeks and 17 patients with AD who did not receive the drug. Profile of Mood States (POMS) scores were used to measure several types of mental stress. Severity of AD was evaluated using the SCORAD Index, and the patients' level of stress and sleeping status were evaluated using visual analogue scales. RESULTS: Before TC administration, all scores were markedly different in the 37 patients compared with 37 normal healthy controls matched for age and gender. POMS scores for tension-anxiety (T-A) and the SCORAD Index decreased significantly in patients who received TC, but did not change significantly in the untreated patients. The two groups had significantly different treatment responses based on changes in T-A scores. There was a significant correlation between changes in the T-A score and SCORAD Index. CONCLUSION: These data suggest that anxiolytic drugs such as 5-HT(1A) agonists are useful in the clinical management of stress-associated aggravation of AD. Inhibition of stress-induced mast cell degranulation may be one of the mechanisms underlying the clinical efficacy.

Cutaneous immunological activation elicited by a low-fluence pulsed dye laser.

BACKGROUND: Three years ago, the nonablative wrinkle reduction laser (a 585-nm laser, Chromogenex V3; Chromogenex Light Technologies, Llanelli, U.K.) was developed, and there have already been several reports about its clinical effectiveness. The Chromogenex V3 laser has also been reported to be effective in treating acne and atopic dermatitis. These results suggest that the Chromogenex V3 laser has some immunological role. In this study, we investigated immunological changes elicited by laser irradiation at the ultrastructural level and by analysis of interleukin (IL)-2 and IL-4 mRNA in skin homing T lymphocytes. MATERIALS AND METHODS: Eight healthy adult volunteers (mean age 56.3 years, range 25-66 years) were recruited for this study. Ultrastructural analysis was done 3 h after the laser irradiation, as well as 1 day, 3 days, 1 week, 2 weeks, 4 weeks and 5 weeks later. IL-2 and IL-4 mRNAs in skin homing T cells cultured for 6 weeks were semiquantitatively measured using reverse transcriptase-polymerase chain reaction. RESULTS: Ultrastructural observations revealed that at 3 h after laser therapy, neutrophils, monocytes and mast cells could already be seen in the extravascular dermis. These dermal acute inflammatory changes were observed also at 1 week after laser treatment. Two weeks after laser treatment, the capillaries showed an almost normal structure. Four weeks after laser treatment, many lymphocytes and fibroblasts were observed. The numbers of these lymphocytes increased further at 5 weeks after the laser treatment. One week after the laser irradiation, all subjects were positive for IL-2 mRNA and for IL-4 mRNA. The level of IL-4 mRNA was larger compared with that of IL-2 mRNA in all subjects. CONCLUSION: The Chromogenex V3 is a 585-nm visible light laser, and it may affect the skin not only by selective photothermolysis but also by direct cutaneous immunological activation.

Identification and characterization of novel peroxisome proliferator-activated receptor-gamma (PPAR-gamma) transcriptional variants in pig and human.

The peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a member of the steroid/thyroid/retinoid receptor superfamily, and is primarily expressed in fat tissue. To date, two major PPAR-gamma isoforms have been identified in pig, PPAR-gamma1 and PPAR-gamma2. Porcine PPAR-gamma1a consists of two leader exons, designated A1 and A2, followed by six exons containing the open reading frame. Here, we report the isolation and characterization of three novel PPAR-gamma1 transcripts. PPAR-gamma1b is derived from exon A1, with exon A2 spliced out. PPAR-gamma1c and PPAR-gamma1d are derived from the new exon, A', containing exon A2 (gamma1c) or without exon A2 (gamma1d). Based on PCR analysis of PAC clones that included sequences from the 5'-untranslated region of the PPAR-gamma gene, the new A' exon is located between the known exons A1 and A2. We also isolated the human homologue to exon A', as well as the two new PPAR-gamma1c and -gamma1d splice variants, from human adipose tissue. Studies of the expression of porcine PPAR-gamma by real time reverse transcription-polymerase chain reaction analysis show that transcripts derived from exon A1 were not expressed at significantly different levels in visceral fat (lamina subserosa) or subcutaneous fat (back fat, inner and outer layer). In contrast, exon A'-derived transcripts were expressed at progressively higher levels in the inner and outer layers of subcutaneous fat than in visceral fat. The same expression pattern was also observed for PPAR-gamma2. We hypothesize that there are three promoters, which differentially regulate PPAR-gamma1 and PPAR-gamma2 gene expression, depending on the specific localization of the fat tissue.

cDNA cloning, mapping and polymorphism of the porcine Rhesus (RH) gene.

The Rhesus (Rh) gene superfamily in humans and mice contains four independent genes, RH, RHAG, RHBG, and RHCG/GK. Heretofore, only the RHBG cDNA has been cloned in pig. We have isolated the porcine RH cDNA; its complete open reading frame of 1269 nucleotides encoded 423 amino acids. Porcine RH protein shared 67.6% amino acid identity with bovine RH, 61.0% with human RhCE and 60.8% with human RhD. The RT-PCR revealed RH transcripts in the spleen and bone marrow, but not in the heart, kidney, or lung. In RH intron 4, a deletion of 17 nucleotides distinguished the shorter allele (allele 1) from the longer. As determined in 115 unrelated pigs from five breeds - Landrace (L, n = 23), Large White (LW, n = 28), Duroc (D, n = 24), Hampshire (H, n = 20) and Piétrain (n = 20) - allele 1 frequencies were 1.0 (L, H), 0.77 (LW), 0.70 (P) and 0.25 (D). Somatic cell hybrid mapping localized the porcine RH and RHBG genes to pig chromosomes 6q22-q23 and 4q21-q22, respectively. Genetic mapping suggested RH-(FUT1, S, GPI, EAH, A1BG)-PGD as the most probable locus order. Sequence homology, mapping data, and haematopoietic tissue expression suggest that this cDNA may indeed encode the porcine RH homologue.

Reactivity of autoantibodies of autoimmune hemolytic anemia with recombinant rhesus blood group antigens or anion transporter band3.

The specificity of autoantibodies in autoimmune hemolytic anemia (AIHA) has been studied using the serological procedure and immunoprecipitation technique with rare phenotype red cells. We attempted to analyze specificity using recombinant rhesus (Rh) blood group and band3 antigens expressed on erythroleukemic cell lines, KU812E. The autoantibody eluates were isolated by the acid elution procedure from the red cells of 20 AIHA patients. The recombinant Rh antigens, RhD, cE, ce, CE, and chimera antigens CE-D and D-CE, were obtained by retroviral cDNA transduction into KU812E cells, and the cell line expressing the antigens was cloned. Band3 cDNA was also obtained and introduced into KU812E and cloned KU812 expressing RhcE. The reactivities of AIHA eluates with recombinant Rh and band3 antigens were studied by flow cytometry. Fifteen eluates reacted with at least one of the RhcE, ce, or CE antigens, and four eluates reacted with RhD. Seven eluates with strong Rh specificity were studied further using chimera antigen. Five eluates showed reduced or lost reactivity, although two eluates reacted identically with the chimera antigens as wild type. These results indicated that conformational epitopes constituted by RhD or CE specific exofacial peptide loops are important for autoantibodies in most cases. Seven eluates reacted with band3, five exclusively. The coexpression study of RhcE and band3 did not enhance the expression of either antigen nor the reactivity with patient eluates, indicating that association of Rh and band3 was not involved in the appearance of autoantigen.

Molecular characterization of weak D phenotypes by site-directed mutagenesis and expression of mutant Rh-green fluorescence protein fusions in K562 cells.

BACKGROUND AND OBJECTIVES: Mutations detected in 161 weak D samples from Caucasians have been classified into 16 types. Because flow cytometry using monoclonal anti-D antibodies (mAbs) has shown that weak D red cells display type-specific antigen density, these mutations in transmembranous regions have been assigned weak D phenotypes. The present study attempts to confirm or refute this assignment. MATERIALS AND METHODS: We amplified DNA from four Japanese weak D samples using the polymerase chain reaction (PCR), and directly sequenced the amplified DNA. Using site-directed mutagenesis, we constructed three vectors expressing mutant RHDs-- G212C, V270G (weak D type 1) and G358A (type 2)--in K562 cells. The expression of RhD antigens was examined by flow cytometry using mAbs. RESULTS: A new mutation resulting in a conversion at amino acid residue 212 (Gly to Cys) was detected in a Japanese weak D sample. K562 cells transduced with mutant RhD cDNA reacted weakly in a type-specific manner with mAbs. CONCLUSIONS: The mutations--G212C (new weak D type), V270G (weak D type 1) and G358A (type 2)-- in transmembranous regions had obvious effects on the D epitopes recognized by mAbs. The results of this study provide direct evidence that these mutations can account for weak D phenotypes.

The analysis of nucleotide substitutions, gaps, and recombination events between RHD and RHCE genes through complete sequencing.

We determined the entire nucleotide sequences of all introns within the RHD and RHCE genes by amplifying genomic DNA using long PCR methods. The RHD and RHCE genes were 57,295 and 57,831 bp in length, respectively. Aligning both genes revealed 138 gaps (insertions and deletions) below 100 bp, 1116 substitutions in all introns and all exons (coding region), and 5 gaps of over 100 bp. Homologies (%) between the RH genes were 93.8% over all introns and coding exons and 91.7% over all exons and introns. Various short tandem repeats (STRs) and many interspersed nuclear elements were identified in both genes. The proportions of Alu sequences in the RHD and RHCE genes were 25.9 and 25.7%, respectively and these Alu sequences were concentrated in several regions. We confirmed multiple recombinations in introns 1 and 2. Such multiple recombination, which probably arose due to the concentrations of Alu sequences and the high level of the homology (%), is one of most important factors in the formation and evolution of RH gene. The variability of the Rh system may be generated because of these features of RH genes. Apparent mutational hotspots and regions with low of K values (the numbers of substitutions per nucleotide site) caused by recombinations as well as true mutational hotspots may be found in human genome. Accordingly, in searching for and identifying single nucleotide polymorphisms (SNPs) especially in noncoding regions, apparent mutational hotspots and areas of low K values by recombination should be noted since the unequal distribution of SNPs will reduce the power of SNPs as genetic maker. Combining the complete sequences' data of both RH genes with serological findings will provide beneficial information with which to elucidate the mechanism of recombination, mutation, polymorphism, and evolution of other genes containing the RH gene as well as to analyze Rh variants and develop new methods of Rh genotyping.

Cloning and characterization of erythroid-specific DNase I-hypersensitive site in human rhesus-associated glycoprotein gene.

Rhesus-associated glycoprotein is a critical co-factor in the expression of rhesus blood group antigens. We identified and cloned an erythroid-specific major DNase I-hypersensitive site located about 10 kilobases upstream from the translation start site of the RHAG gene. A short core enhancer sequence of 195 base pairs that corresponded with the major hypersensitive site and possessed position- and orientation-independent enhancer activity in K562 cells. In vitro DNase I footprint analysis revealed four protected regions in the core enhancer; two GATA motifs, an Ets-like motif and an unknown motif. The GATA motifs bound GATA-1 and mutagenesis analysis revealed that the proximal one is critical for the enhancing activity. Homology plot analysis using the 5' sequence of the mouse RHAG gene revealed four homologous stretches and multiple insertions of repetitive sequences among them; four LINE/L1 and four Alu in the human and as well as one LINE/L1 and one LTR/MaLR in the mouse gene. The highly conservative enhancer region was flanked by SINE and LINE/L1 in both species. These results suggest that the 5'-flanking sequence of RHAG gene is a preferable target sequence for retroviral transposition and that the enhancer was inserted in the same manner, resulting in the acquisition of erythroid dominant expression.

A Japanese propositus with D-- phenotype characterized by the deletion of both the RHCE gene and D1S80 locus situated in chromosome 1p and the existence of a new CE-D-CE hybrid gene.

In a family study of a Japanese propositus with the D-- phenotype, the serological data of her D-- phenotype and those of her parents were discrepant. Gene analysis of the propositus showed a gross deletion of the RHCE gene and a new rearrangement of RHCE to yield the CE-D-CE hybrid. It was demonstrated that the hybrid CE-D-CE gene consisted of exon 1 from the RHCE gene, followed by exons 3 to 7 from the RHD gene and exons 8 to 10 from the RHCE gene. However, whether or not exon 2 of the RHD or the RHCE gene was contained in the CE-D-CE gene remained unclear. Moreover, spacer analysis between both RH genes and the family study suggested that the D-- gene complex from the paternal and maternal sides consisted of only the CE-D-CE hybrid gene and a single RHD gene, respectively. For the purpose of confirming the parent-child relationship, a paternity test using DNA fingerprint and polymerase chain reaction (PCR) analysis at the D1S80 locus were performed. DNA fingerprints with two kinds of DNA minisatellite probes (33.15 and 33.6) confirmed that the parent-child relationship in the D-- propositus was compatible. However, in the present case, at the D1S80 locus, the PCR product derived from the mother was lacking, thereby negating a parent-child relationship. It is probable that the RH genes and D1S80 locus exist in close proximity, because they are situated in chromosomes 1p 34.3-36.1 and 1p 36.1-36.3, respectively. These data suggested that at the stage of gametogenesis, both the RHCE gene and the D1S80 locus from the maternal side may have been deleted, thereby producing the D-- gene complex.