A glycomics approach to discover novel renal biomarkers in birds by administration of cisplatin and diclofenac to chickens.

Avian species have a unique renal structure and abundant blood flow into the kidneys. Although many birds die due to nephrotoxicity caused by chemicals, there are no early biomarkers for renal lesions. Uric acid level in blood, which is generally used as a renal biomarker, is altered when the kidney function is damaged by over 70%. Therefore, early biomarkers for kidney injury in birds are needed. In humans, glycomics has been at the forefront of biological and medical sciences, and glycans are used as biomarkers of diseases, such as carcinoma. In this study, a glycomics approach was used to screen for renal biomarkers in chicken. First, a chicken model of kidney damage was generated by injection of diclofenac or cisplatin, which cause acute interstitial nephritis (AIN) and acute tubular necrosis (ATN), respectively. The nephrotoxicity levels were determined by a blood chemical test and histopathological analysis. The plasma N-glycans were then analyzed to discover renal biomarkers in birds. Levels of 14 glycans increased between pre- and post administration in kidney-damaged chickens in the diclofenac group, and some of these glycans had the same presumptive composition as those in human renal carcinoma patients. Glycan levels did not change remarkably in the cisplatin group. It is possible that there are changes in glycan expression due to AIN, but they do not reflect ATN. Although further research is needed in other species of birds, glycans are potentially useful biomarkers for AIN in avian species.

Diffusion-weighted MRI as a potential imaging biomarker reflecting the metastatic potential of upper urinary tract cancer.

OBJECTIVE: To evaluate the role of diffusion-weighted MRI (DW-MRI) as an imaging biomarker for upper urinary tract cancer (UUTC) that has already metastasized or will metastasize soon. METHODS: 61 patients clinically diagnosed with UUTC were prospectively enrolled in this study. All the patients underwent MRI, including DW-MRI, prior to any interventions. Correlations between apparent diffusion coefficient (ADC) and other clinicopathological variables, including metastasis-free survival, were analysed. RESULTS: Median follow-up period was 938 days. Of the 61 patients, 12 had any metastases at the initial diagnosis. 11 patients developed metastases during the follow-up period. These 23 patients were categorized as "Metastatic". Of the remaining 38 patients, 35 with a follow-up period longer than 400 days were categorized as "Localized". ADC was significantly lower in the Metastatic category than in the Localized (p = 0.0002) category. Multivariate analysis of pre-operative variables identified ADC (cut-off value, 1.08 × 10(-3) mm(2) s(-1)) and clinical T stage based on T2 weighted MRI as an independent predictive factor of metastatic UUTC. 46 patients without any metastases during the initial diagnosis were stratified into a high-risk group (16 patients with low ADC and clinical T3-4) and a low-risk group (30 patients with high ADC or clinical Ta-2). The 3-year metastasis-free survivals were 45% and 93%, respectively. CONCLUSION: In the current study, UUTC with lower ADC value is more likely to have metastatic potential. Incorporating ADC with clinical T stage helps to differentiate metastatic UUTC at the initial diagnosis. ADVANCES IN KNOWLEDGE: DW-MRI is a potential imaging biomarker reflecting metastatic propensity of UUTC.

Analyses of laboratory data and establishment of reference values and intervals for healthy elderly people.

OBJECTIVE: Protein-energy malnutrition is a common disorder in the elderly. Although serum albumin is commonly used as a nutritional marker, data is lacking on serum albumin levels in the elderly. The purpose of this study was to determine whether serum albumin levels decrease with advancing age and to establish reference value and interval of laboratory data for elderly people (75 years and over). PARTICIPANTS: Blood samples from 13821 healthy people, 42064 outpatients, and 15959 inpatients were collected during 2008. Blood from 127 of our nutrition support team (NST) patients was also collected during August 2006 and May 2009, and analyzed. MEASUREMENTS: Serum albumin, hemoglobin, total cholesterol levels and lymphocyte count were determined. We analyzed the change in each parameter in accordance with age, compared the data for elderly people with younger people, and established new reference values. Clinical outcomes were examined depending on the improved reference values. RESULTS: Albumin was lower in older persons than in younger persons. The estimated reference value and interval were 42 (48-36) g/l in older persons and was much lower in NST patients. Hemoglobin was decreased while cholesterol and lymphocyte count were not changed in older persons: all were markedly decreased in NST patients. Terms of hospital stay were significantly longer and mortality rates were significantly higher in older persons, comparing from above to below using a new reference value of albumin (36 g/l). CONCLUSIONS: The serum albumin level decreases with advancing age, but it was maintained to some extent in healthy older people. Serum albumin levels related to the clinical outcome. Hemoglobin and cholesterol levels and lymphocyte count were all lower in NST patients. These measurements may be valuable markers of nutritional status and can help in guiding the need for nutritional support.

New treatment of free-radical scavenger in adrenoleukodystrophy.

WHAT IS KNOWN AND OBJECTIVE: Adrenoleukodystrophy (ALD) is an X-linked disorder and characterized by the accumulation of saturated very long-chain fatty acids. Treatment is still unsatisfactory. Our objective is to report on the effect of the free-radical scavenger, edaravone, in a patient with ALD. CASE SUMMARY: The patient was given edaravone intravenously twice. D-ROM in cerebral spinal fluid decreased dramatically, and a shortening of neuronal transmission time as estimated on somatosensory evoked potential was observed. After terminating the treatment, his symptoms progressively reappeared. WHAT IS NEW AND CONCLUSION: This is the first report of the use of edaravone in ALD. The drug is apparently effective in improving symptoms of ALD and should be evaluated more formally.

Relationship between transcardiac gradient of endothelin-1 and left ventricular remodelling in patients with first anterior myocardial infarction.

Aims To evaluate whether plasma endothelin-1 (ET-1) is extracted or produced through the heart in patients with acute myocardial infarction (AMI), and the relationship between transcardiac extraction of plasma ET-1 and left ventricular (LV) remodelling. Methods and results We measured the plasma level of ET-1 in the aortic root (Ao) and coronary sinus (CS) in 48 consecutive patients, who received successful revascularization and enalapril, for a first anterior AMI. In the acute phase the plasma ET-1 level was significantly higher both in the Ao and the CS compared to the control subjects. However, the plasma ET-1 level was significantly lower in the CS than in the Ao in the acute phase and after 1 month. There were significant correlations between transcardiac extraction of ET-1 in the acute phase and LV ejection fraction and LV end-diastolic volume index (LVEDVI) after 1 month. Stepwise multivariate analysis showed that maximal creatine phosphokinase and transcardiac extraction of plasma ET-1 during the acute phase were independently and positively correlated with the absolute change in LVEDVI after 1 month. Conclusions These results indicate that elevated circulating ET-1 is extracted through the heart in patients with a first anterior AMI and that the extracted ET-1 plays a significant role in modulating post-infarct LV remodelling.

The upr-1 gene encodes a catalytic subunit of the DNA polymerase zeta which is involved in damage-induced mutagenesis in Neurospora crassa.

The upr-1 mutant was one of the first mutagen-sensitive mutants to be isolated in Neurospora crassa. However, the function of the upr-1 gene has not yet been elucidated, although some genetic and biochemical data have been accumulated. In order to clone the upr-1 gene, we performed a chromosome walk from the mat locus, the closest genetic marker to upr-1 for which a molecular probe was available, towards the centromere, and a chromosomal contig of about 300-400 kb was constructed. Some of these clones complemented the temperature sensitivity of the un-16 mutation, which is located between mat and upr-1. The un-16 gene was sequenced, and localized in the MIPS Neurospora crassa genome database. We then searched the regions flanking un-16 for homologs of known DNA repair genes, and found a gene homologous to the REV3 gene of budding yeast. The phenotype of the upr-1 mutant is similar to that of the yeast rev3 mutant. An ncrev3 mutant carrying mutations in the N. crassa REV3 homolog was constructed using the RIP (repeat-induced point mutation) process. The spectrum of mutagen sensitivity of the ncrev3 mutant was similar to that of the upr-1 mutant. Complementation tests between the upr-1 and ncrev3 mutations indicated that the upr-1 gene is in fact identical to the ncrev3 gene. To clarify the role of the upr-1 gene in DNA repair, the frequency of MMS and 4NQO-induced mutations was assayed using the ad-8 reversion test. The upr-1 mutant was about 10 times less sensitive to both chemicals than the wild type. The expression level of the upr-1 gene is increased on exposure to UV irradiation in the uvs-2 and mus-8 mutants, which belong to postreplication repair group, as well as in the wild type. All these results suggest that the product of the upr-1 gene functions in damage-induced mutagenesis and DNA translesion synthesis in N. crassa.

Diabetic ketoacidosis in a case of pheochromocytoma.

A 31-year-old woman was admitted to our hospital because of diabetic ketoacidosis (DKA). Ultrasound sonography revealed the existence of the left adrenal tumor and endocrinological examinations established a diagnosis of pheochromocytoma. She had been healthy and there was no evidence for gestational diabetes in her personal history. Characteristic features were not found in her tumor size and the catecholamine levels as compared with typical cases of pheochromocytoma. An overwhelming secretion of catecholamine might suppress insulin secretion, as evidenced by the improvement after the resection of the tumor. However, a significant insulin resistance continued after tumor resection. Obesity and the heterozygosity of beta3-adrenergic receptor gene (Try64Arg) might play a role in insulin resistance, which resulted in DKA at least in part. Literature survey revealed four cases of DKA in the patients with pheochromocytoma including ours, three of which were Japanese. Pancreatic capacity to secrete insulin has been reported to be less than Caucasians, which might be another reason for DKA. Thus, we speculate that both suppressed insulin secretion and insulin resistance deteriorated by obesity or other factor(s) such as abnormality in beta3 adrenergic receptor probably depress beta-cell function resulting in abnormal metabolic imbalance such as DKA.

Regulation of c-Myb activity by tumor suppressor p53.

Heat shock proteins (HSPs) act as chaperones and play important roles during cellular proliferation and apoptosis. Heat shock factors (HSFs) mediate transcriptional induction of HSP genes. Among multiple heat shock transcription factors (HSFs) in vertebrates, HSF3 is specifically activated in unstressed proliferating cells by direct binding to the c-myb proto-oncogene product (c-Myb). Since c-Myb has an important role in cellular proliferation, this regulatory pathway suggests a link between the events of cellular proliferation and the stress response. The c-Myb-induced activation of HSF3 is negatively regulated by the p53 tumor suppressor protein. p53 directly binds to HSF3 and blocks the interaction between c-Myb and HSF3. In addition, p53 stimulates the degradation of c-Myb, which is, at least partly, mediated by induction of Siah in certain types of cells. Thus, c-Myb and p53 regulate the expression of HSPs via HSF3 in opposite ways.

Constitutive c-Myb amino-terminal phosphorylation and DNA binding activity uncoupled during entry and passage through the cell cycle.

The c-myb gene encodes a transcription factor that is central to hematopoietic cell growth. Phosphorylation of c-Myb by casein kinase 2 (CK2) at serines 11 and 12 has been variously implicated in the regulation of DNA binding. However, it is unclear when c-Myb phosphorylation at serines 11 and 12 occurs during the cell cycle and how this is regulated. We generated specific antisera that recognize phosphoserines 11 and 12 of c-Myb. C-Myb protein levels, extent of CK2 phosphorylation and DNA binding were then monitored following mitogenic stimulus and passage through the cell cycle in normal peripheral T-cells and the T leukemia cell line CCRF-CEM. We found that endogenous c-Myb is constitutively phosphorylated at serines 11 and 12. The amount of phosphorylated c-Myb correlates with DNA binding activity in cycling CEM cells but not upon entry of T-cells into the cell cycle. Exogenous expression of c-Myb with substitutions of serines 11 and 12 with glutamic acid or alanine had no effect on the transactivation of a c-Myb responsive reporter. These data strongly suggest that c-Myb is constitutively phosphorylated on serines 11 and 12 by CK2 or like activity and is not regulated during the cell cycle.

A Neurospora double-strand-break repair gene, mus-11, encodes a RAD52 homologue and is inducible by mutagens.

We isolated a Neurospora crassa cDNA that encodes a Rad52 homologue (ncRAD52) by PCR, using degenerate primers. RFLP mapping demonstrated that the cloned gene is located close to the ro-4 locus on the right arm of linkage group V (LGVR). In a second experiment, we used sib selection to identify a cosmid clone containing the mus-11 gene in a N. crassa genomic library. Fine-scale mapping of the mus-11 mutant showed the gene order on LGVR near ro-4 to be: ad-7 - (9.5 mu) - pab-2 (7.8 mu) - mus-11 - (3.7 mu) - inv. The nucleotide sequence of the mus-11 gene matched that of the ncRAD52 cDNA. Thus, the mus-11 gene encodes the Rad52 homologue. The deduced amino acid sequence of the MUS11 protein shows 32.0% and 27.5% overall identity to the Schizosaccharomyces pombe Rad22 protein and the human hRad52 protein, respectively, and a higher level of identity (55-66%) within the conserved N-terminal region (141 residues). The MUS11 protein shows homology to Rad52 from budding yeast only within the N-terminal region (53.2% identity over 141 amino acids) which is conserved among Rad52 homologues. Yeast two-hybrid analysis reveals that the MUS11 protein binds to both the MEI-3 protein, a Rad51 homologue, and to itself in vivo. An ncRAD52 mutant obtained by the RIPping procedure showed the same sensitivity as the original mus-11 mutant to the following mutagens and chemicals: UV light, 4NQO (4-nitroquinoline 1-oxide), MMS (methyl methanesulfonate), EMS (ethyl methanesulfonate), MNNG (N-methyl-N'-nitro-N-nitrosoguanidine), TBHP (tert-butyl hydroperoxide), HU (hydroxyurea) and histidine. Unlike the RAD52 transcript in Saccharomyces cerevisiae, the mus-11 transcript could not be detected in mycelium under normal growth conditions, but expression of the gene was induced by UV irradiation or treatment with MMS.

Characterization of the Neurospora crassa mus-25 mutant: the gene encodes a protein which is homologous to the Saccharomyces cerevisiae Rad54 protein.

Characterization of the Neurospora crassa mus-25 mutant suggests that it is defective in recombination repair and belongs to the uvs-6 epistasis group. It shows a high sensitivity to the alkylating agents methyl methanesulfonate (MMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), but not to UV radiation. It is barren (i.e. does not produce ascospores) in homozygous crosses. The frequency of MMS-induced mutations at the ad-3 loci is approximately three times higher than in the wild type. The ratio of homologous to nonhomologous integration of the pMTR::HYG plasmid is much lower than in wild type. The mus-25 mutant is epistatic to the mei-3 mutant for MMS sensitivity. mei-3, which is a homololog of the Saccharomyces cerevisiae gene RAD51, is a member of the uvs-6 epistasis group which contains several genes that are homologous to recombination repair genes in other organisms. The mus-25 gene was cloned by identifying a genomic DNA fragment which complements the MMS sensitivity of the mutant. The amino acid sequence deduced from the cloned DNA showed a high degree of homology to the Rad54 protein, which is involved in recombinational repair in S. cerevisiae. Comparison of the nucleotide sequences of the genomic and cDNAs of the mus-25 gene revealed an ORF of 2505 bp with a single 118-bp intron beginning immediately after the second nucleotide of the AUG start codon. The molecular weight of the deduced gene product was 93.5 kDa. The transcript level was raised within 60 min after UV irradiation or MMS treatment, as also observed for the expression of the other N. crassa recombinational repair genes, suggesting the existence of a common mechanism which induces expression of the recombinational repair genes in response to DNA damage.

p53 suppresses the c-Myb-induced activation of heat shock transcription factor 3.

Expression of heat shock proteins (HSPs) is controlled by heat shock transcription factors (HSFs). Vertebrates express multiple HSFs whose activities may be regulated by distinct signals. HSF3 is specifically activated in unstressed proliferating cells by direct binding to the c-myb proto-oncogene product (c-Myb), which plays an important role in cellular proliferation. This suggests that the c-Myb-induced HSF3 activation may contribute to the growth-regulated expression of HSPs. Here we report that the p53 tumor suppressor protein directly binds to HSF3 and blocks the interaction between c-Myb and HSF3. In addition, p53 stimulates the degradation of c-Myb through a proteasome-dependent mechanism, which is, at least partly, mediated by induction of Siah in certain types of cells. Induction of p53 by a genotoxic reagent in DT40 cells disrupts the HSF3-c-Myb interaction and down-regulates the expression of certain HSPs. Mutated forms of p53 found in certain tumors did not inhibit c-Myb-induced HSF3 activation. The regulation of HSF3 activity by c-Myb and p53 sheds light on the molecular events that govern HSP expression during cellular proliferation and apoptosis.

Detection of DNA restriction fragment polymorphisms in Streptococcus equi.

Large-restriction-fragment (LRF) polymorphisms in Streptococcus equi (S equi subspecies equi) were studied by pulsed-field gel electrophoresis. Five or six chromosomal fragments of between 194 and 915 kb were separated by digestion with the restriction endonuclease Notl. All 20 isolates of S equi, including 12 from independent Japanese outbreaks, four from independent American outbreaks, two from a single Irish outbreak, us vaccine strain F43, and type strain NCTC 9682 were successfully typed. Seven distinctive, reproducible and stable types were identified. The 12 Japanese isolates collected between 1992 and 1998 were of LRF type II suggesting that they were derived from the same source. The remaining eight isolates were of six types. The results indicate that LRF typing should be a useful technique for investigating the source and transmission of S equi.

[In vitro and in vivo activities of cefpodoxime proxetil against penicillin-resistant Streptococcus pneumoniae].

We evaluated in vitro and in vivo activities of cefpodoxime proxetil (CPDX-PR) in comparison with other oral beta-lactams, cefdinir (CFDN), cefditoren pivoxil (CDTR-PI), and faropenem (FRPM), against penicillin-susceptible and -resistant Streptococcus pneumoniae. In vitro activities (MICs) of CPDX, CFDN, CDTR, and FRPM against clinical isolates, penicillin-susceptible S. pneumoniae (PSSP: MIC of penicillin G, < or = 0.063 microgram/ml), penicillin-intermediate S. pneumoniae (PISP: MIC of penicillin G, 0.125-1 microgram/ml), and penicillin-resistant S. pneumoniae (PRSP: MIC of penicillin G, > or = 2 micrograms/ml), were tested by an agar dilution method. The MIC80s of CPDX against 27 PSSP strains, 23 PISP strains, and 23 PRSP strains were 0.032, 1, and 8 micrograms/ml, respectively, which were superior to or equal to those of CFDN (0.063, 4, and 8 micrograms/ml) and were inferior to those of CDTR (0.016, 0.5, and 1 microgram/ml) and FRPM (< or = 0.008, 0.25, and 1 microgram/ml). Infection was induced in mice by inoculating with a PRSP clinical isolate, 9605 or 9601 (serotype 6), or 10692 (serotype 19), through the nares of male ddY mice into the lungs. The mice were treated with drugs with doses of 2-50 mg/kg at 18, 26, 42, and 50 hours after the infection. Viable cell numbers in the lungs and blood were assayed at 66 hours after the infection. The efficacy of each drug was dose-dependent. CPDX-PR showed the most potent in vivo efficacy among the drugs tested against the infections caused by the PRSP strains. MICs of the drugs against PRSP 9605, 9601, and 10692 were as follows: CPDX, 4, 4 and 2 micrograms/ml; CFDN, 16, 16, and 4 micrograms/ml; CDTR, 1, 1, and 0.5 microgram/ml; and FRPM, 1, 0.5, and 0.5 microgram/ml, respectively. Thus, CPDX-PR showed a stronger in vivo activity than that expected from the MICs of CPDX. This was probably caused by the pharmacokinetic advantage of CPDX over the other drugs used in this study.

High-resolution structure of the conger eel galectin, congerin I, in lactose-liganded and ligand-free forms: emergence of a new structure class by accelerated evolution.

BACKGROUND: Congerin I is a member of the galectin (animal beta-galactoside-binding lectin) family and is found in the skin mucus of conger eel. The galectin family proteins perform a variety of biological activities. Because of its histological localization and activity against marine bacteria and starfish embryos, congerin I is thought to take part in the eels' biological defense system against parasites. RESULTS: The crystal structure of congerin I has been determined in both lactose-liganded and ligand-free forms to 1. 5 A and 1.6 A resolution, respectively. The protein is a homodimer of 15 kDa subunits. Congerin I has a beta-sheet topology that is markedly different from those of known relatives. One of the beta-strands is exchanged between two identical subunits. This strand swap might increase the dimer stability. Of the known galectin complexes, congerin I forms the most extensive interaction with lactose molecules. Most of these interactions are substituted by similar interactions with water molecules, including a pi-electron hydrogen bond, in the ligand-free form. This observation indicates an increased affinity of congerin I for the ligand. CONCLUSIONS: The genes for congerin I and an isoform, congerin II, are known to have evolved under positive selection pressure. The strand swap and the modification in the carbohydrate-binding site might enhance the cross-linking activity, and should be the most apparent consequence of positive selection. The protein has been adapted to functioning in skin mucus that is in direct contact with surrounding environments by an enhancement in cross-linking activity. The structure of congerin I demonstrates the emergence of a new structure class by accelerated evolution under selection pressure.

PPAR gamma mediates high-fat diet-induced adipocyte hypertrophy and insulin resistance.

Agonist-induced activation of peroxisome proliferator-activated receptor gamma (PPAR gamma) is known to cause adipocyte differentiation and insulin sensitivity. The biological role of PPAR gamma was investigated by gene targeting. Homozygous PPAR gamma-deficient embryos died at 10.5-11.5 dpc due to placental dysfunction. Quite unexpectedly, heterozygous PPAR gamma-deficient mice were protected from the development of insulin resistance due to adipocyte hypertrophy under a high-fat diet. These phenotypes were abrogated by PPAR gamma agonist treatment. Heterozygous PPAR gamma-deficient mice showed overexpression and hypersecretion of leptin despite the smaller size of adipocytes and decreased fat mass, which may explain these phenotypes at least in part. This study reveals a hitherto unpredicted role for PPAR gamma in high-fat diet-induced obesity due to adipocyte hypertrophy and insulin resistance, which requires both alleles of PPAR gamma.

Characterization of a Neurospora crassa photolyase-deficient mutant generated by repeat induced point mutation of the phr gene.

We produced a photolyase-deficient mutant by repeat induced point mutation using the Neurospora crassa photolyase gene cloned previously. This mutation identified a new gene, phr, which was mapped on the right arm of linkage group I by both RFLP mapping and conventional mapping. To investigate the relationship between photoreactivation and dark repair processes, especially excision repair, double mutants of phr with representative repair-defective mutants of different types were constructed and tested for UV sensitivity and photoreactivation. The results show that the phr mutation has no influence on dark repair. Tests with CPD and TC(6-4) photoproduct-specific antibodies demonstrated that the phr mutant is defective in CPD photolyase and confirmed that there is no TC(6-4) photolyase activity in N. crassa. Furthermore, N. crassa photolyase is not a blue light receptor in the signal transduction that induces carotenoid biosynthesis.

Accelerated evolution in the protein-coding region of galectin cDNAs, congerin I and congerin II, from skin mucus of conger eel (Conger myriaster).

Two cDNAs encoding galectins named congerins I and II from the skin mucus of conger eel (Conger myriaster) were isolated and sequenced. Comparison of the nucleotide sequences of congerins I and II showed that the sequence similarities of the 5' and 3' untranslated regions (86 and 88%, respectively) were much higher than those of the protein-coding region (73%). The numbers of nucleotide substitutions per site (KN) for the untranslated regions are smaller than the numbers of nucleotide substitutions per synonymous site (KS) for the protein coding region. Furthermore, nonsynonymous nucleotide substitutions have accelerated more frequently than synonymous nucleotide substitutions in the protein coding region (KA/KS = 2.57). These results suggest that accelerated substitutions have occurred in the protein-coding regions of galectin genes to generate diverse galectins with different molecular properties. Northern blot analysis showed that both congerins were expressed not only in the skin tissues but also in the stomach of conger eel.