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.

Activation of heat shock transcription factor 3 by c-Myb in the absence of cellular stress.

In vertebrates, the presence of multiple heat shock transcription factors (HSFs) indicates that these factors may be regulated by distinct stress signals. HSF3 was specifically activated in unstressed proliferating cells by direct binding to the c-myb proto-oncogene product (c-Myb). These factors formed a complex through their DNA binding domains that stimulated the nuclear entry and formation of the transcriptionally active trimer of HSF3. Because c-Myb participates in cellular proliferation, this regulatory pathway may provide a link between cellular proliferation and the stress response.

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.

Development of non-insulin-dependent diabetes mellitus in the double knockout mice with disruption of insulin receptor substrate-1 and beta cell glucokinase genes. Genetic reconstitution of diabetes as a polygenic disease.

Non-insulin-dependent diabetes mellitus (NIDDM) is considered a polygenic disorder in which insulin resistance and insulin secretory defect are the major etiologic factors. Homozygous mice with insulin receptor substrate-1 (IRS-1) gene knockout showed normal glucose tolerance associated with insulin resistance and compensatory hyperinsulinemia. Heterozygous mice with beta cell glucokinase (GK) gene knockout showed impaired glucose tolerance due to decreased insulin secretion to glucose. To elucidate the interplay between insulin resistance and insulin secretory defect for the development of NIDDM, we generated double knockout mice with disruption of IRS-1 and beta cell GK genes by crossing the mice with each of the single gene knockout. The double knockout mice developed overt diabetes. Blood glucose levels 120 min after intraperitoneal glucose load (1.5 mg/g body wt) were 108 +/- 24 (wild type), 95 +/- 26 (IRS-1 knockout), 159 +/- 68 (GK knockout), and 210 +/- 38 (double knockout) mg/dl (mean +/- SD) (double versus wild type, IRS-1, or GK; P < 0.01). The double knockout mice showed fasting hyperinsulinemia and selective hyperplasia of the beta cells as the IRS-1 knockout mice (fasting insulin levels: 0.38 +/- 0.30 [double knockout], 0.35 +/- 0.27 [IRS-1 knockout] versus 0.25 +/- 0.12 [wild type] ng/ml) (proportion of areas of insulin-positive cells to the pancreas: 1.18 +/- 0.68%; P < 0.01 [double knockout], 1.20 +/- 0.93%; P < 0.05 [IRS-1 knockout] versus 0.54 +/- 0.26% [wild type]), but impaired insulin secretion to glucose (the ratio of increment of insulin to that of glucose during the first 30 min after load: 31 [double knockout] versus 163 [wild type] or 183 [IRS-1 knockout] ng insulin/mg glucose x 10(3)). In conclusion, the genetic abnormalities, each of which is nondiabetogenic by itself, cause overt diabetes if they coexist. This report provides the first genetic reconstitution of NIDDM as a polygenic disorder in mice.

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.

Transcriptional activation of the c-myc gene by the c-myb and B-myb gene products.

To identify the target genes modulated by the myb gene product (Myb), a co-transfection assay with a Myb expression plasmid was performed. Both c-Myb and B-Myb, another member of the myb gene family, trans-activated the human c-myc promoter. DNAase I footprint analysis using the bacterially expressed c-Myb, identified multiple c-Myb binding sites in the c-myc promoter region. Deletion analysis of the c-myc promoter suggested that some number of Myb binding sites, not a specific Myb binding site, is important for the c-Myb-induced trans-activation of the c-myc promoter. Using the c-myc-chloramphenicol acetyltransferase (CAT) construct as a reporter in a co-transfection assay, the domains of c-Myb required for trans-activation were examined. The functional domains of c-Myb identified using the c-myc promoter were almost the same as those identified previously with the artificial target gene containing Myb binding sites, but unlike the case with the artificial target gene the N-terminal half of the previously identified negative regulatory domains and the C-terminal 136 amino acids were required for the maximal trans-activation of the c-myc promoter. These results indicate that there are some differences in the regulation of Myb-dependent trans-activation in different target genes.

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.

Transformation by carboxyl-deleted Myb reflects increased transactivating capacity and disruption of a negative regulatory domain.

Carboxyl-truncated forms of the product of the c-myb proto-oncogene (Myb) are encoded by the v-myb oncogene, the rearranged c-myb genes of certain murine cell lines and a transforming recombinant c-myb retrovirus. We report here an examination of the abilities of a series of carboxyl deletions of Myb to transform hemopoietic cells. Increasing degrees of truncation resulted in increasing transforming capacity until the deletions removed the region responsible for transactivation by Myb. Because the effects of these deletions on transformation paralleled their previously described effects on the transactivating capacity of Myb but did not correlate with their ability to repress transcription, our results imply that removal of a domain which negatively regulates transactivation is responsible for oncogenic activation of carboxyl-truncated forms of Myb. Moreover, these data support the view that activated forms of myb transform by increasing and/or deregulating the expression of other genes.

Evidence for association between the class I subset of the insulin gene minisatellite (IDDM2 locus) and IDDM in the Japanese population.

Although the shortest (class I) minisatellite (i.e., variable number of tandem repeats [VNTR]) alleles in the 5' region of the insulin gene are positively associated with IDDM in Caucasians, the majority of Japanese are homozygous for class I alleles. Here, we determined the exact length, in number of repeat units (RUs), of class I alleles in Japanese subjects. The distribution of class I alleles in Japanese was trimodal, with peaks located at 32/33, 41, and 44 RUs. The shortest component (i.e., 1S [25-38 RUs]) alleles were significantly increased in the IDDM group compared with the control group (54 vs. 46%; P = 0.040). The 1S/1S genotype was significantly increased in the IDDM patients (34 vs. 20%; P = 0.005; relative risk 2.1). Furthermore, the transmission disequilibrium test of Japanese families with 1S/1M or 1S/1L heterozygous parents confirmed the association of 1S alleles; 17 alleles of 1S and 6 alleles of 1M (39-41 RUs) or 1L (42-44 RUs) were transmitted to affected offspring (P = 0.022). In addition, we found tight linkage of 1S with allele 9 of the tyrosine hydroxylase gene microsatellite and allele (-) of the IGF-II gene Apa I polymorphism, but neither 9 nor (-) alleles were significantly associated with IDDM. The present study suggests that a class I subset may have a role in IDDM susceptibility in Japan. It was revealed that the difference between 1S alleles and 1M or 1L alleles is almost consistently characterized by a sequence variation generated by deletion of two copies of an ACAGGGGTCC CGGGG repeat element, implying that sequence variation of class I alleles may influence disease susceptibility.

Independent control of transcription initiations from two sites by an initiator-like element and TATA box in the human c-erbB-2 promoter.

Transcription of the human c-erbB-2-proto-oncogene starts mainly at two sites, nucleotide positions +1 and -69. The present studies have identified an initiator-like element that specifies the position of transcription initiation at position -69. This initiator-like element contains six GGA repeats and is located just downstream from the transcription start site between positions -68 and -45. In addition, both in vitro and in vivo studies indicated that transcription initiation at position +1 is specified by a TATA box 25 bp upstream from the transcription startpoint. Thus, initiation at two sites in the c-erbB-2 promoter is controlled independently by the initiator-like element and the TATA box.

Phenotypic discordance in monozygotic twins with 22q11.2 deletion.

We report on male monozygotic twins with 22q11.2 deletion and discordant phenotypes. The twins had twin-to-twin transfusion syndrome. Twin 1, the smaller of the pair, had Tetralogy of Fallot, a characteristic facial appearance, swallowing dysfunction, anal atresia, short stature, and mental retardation, whereas twin 2 had a characteristic facial appearance but no other signs of the 22q11 deletion syndrome. Fluorescence in situ hybridization analysis showed a microdeletion on chromosome 22q11.2 in both twins. Zygosity analysis gave a probability of monozygosity greater than 99.999%. These observations indicate that environmental factors or postzygotic events play a role in the phenotypic variability in the twins.

Uptake of remnant like particles (RLP) in diabetic patients from mouse peritoneal macrophages.

To investigate whether the remnant like particles (RLP), separated from serum by an immunoaffinity gel mixture of anti-apo B-100 and apo A-I monoclonal antibodies, are relevant to the initiation or progression of atherosclerosis, the incorporation of RLP into mouse macrophages was studied using histochemical and biochemical techniques. Remnant lipoproteins such as RLP are reported to contain a large quantity of chyloniron and very low density lipoprotein (VLDL) remnants, especially in diabetic patients. The RLP separated from the sera of 32 diabetic patients were found to be predominantly taken up into macrophages harvested from mouse abdominal cavities by the staining method applying oil red O. Furthermore, using 14C-oleate to prove the uptake of lipoproteins by macrophages, the uptake of RLP-VLDL, a VLDL fraction of RLP by ultracentrifugation, was the next highest to that of the oxidized LDL, which suggests that RLP-VLDL is also aggressively taken up by macrophages. The degree of uptake of RLP-VLDL by macrophages was positively correlated with HbA1c of these diabetic patients (r = 0.556, p < 0.01), irrespective of the ways of the treatment of diabetes. In conclusion, RLP can contribute to the foaming of macrophages, which in turn may explain the acceleration of atherosclerosis in diabetic patients.

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.

The role of insulin in coronary atherosclerosis.

In 197 patients with coronary artery disease (CAD) who underwent coronary angiography, 75 g oral glucose tolerance test (OGTT) was performed, measuring plasma glucose(PG) and plasma insulin (IRI) at 4 time points (0, 30, 60 and 120 min). Subjects were separated into two groups by their insulinogenic index (I.I. = delta IRI/delta PG from 0 up to 30 min), 99 cases with good insulin response (I.I. > or = 0.4) and 98 cases with poor insulin response (I.I. < 0.4). Only two subjects were diabetic in good insulin response, while 37 were diabetic in poor insulin response. The severity of coronary atherosclerosis was expressed as a coronary index (CI), calculated according to Balcon's method. Fasting PG and the sum of PG were significantly higher in the latter group, while the sum of IRI was significantly lower. CI was not significantly different, however. In the group with good insulin response, the severity of CAD was significantly correlated to fasting IRI (n = 99, r = -0.387, P < 0.02), but, there was no such relationship in the other group. We conclude that hyperinsulinemia might be a risk factor for ischemic heart disease, but in diabetics it is difficult to link the relationship between fasting IRI and CI.

The relationship between early diabetic nephropathy and control of plasma glucose in non-insulin-dependent diabetes mellitus. The effect of glycemic control on the development and progression of diabetic nephropathy in an 8-year follow-up study.

To clarify the relationship between early diabetic nephropathy and the glycemic control in non-insulin-dependent diabetes mellitus (NIDDM) without hypertension, excretion of urinary albumin was studied retrospectively for 8 years. The patients with early diabetic nephropathy were divided into two groups according to the initial urinary albumin index (UAI: mg/g.creatinine), namely, a normoalbuminuric (less than 15 mg/g.creatinine) and a microalbuminuric group (15 < or = UAI < 200 mg/g.creatinine). Comparisons of changes in UAI were made between good (HbA1 < 9.0% and fasting plasma glucose (FPG) < 140 mg/100 mL throughout the observation period) and poor glycemic control groups after 4 and 8 years. In the patients with normoalbuminuria at the initial determination, five of 11 patients (45.5%) with good glycemic control and 14 of 22 patients (63.6%) with poor glycemic control became microalbuminuric after 8 years, respectively (p < 0.05). In the microalbuminuric patients, five of ten patients (50%) with poor glycemic control became macroalbuminuric (UAI > or = 200 mg/g.creatinine), although only one case worsened of five patients with good glycemic control (p < 0.05). In conclusion, the development or progression of early diabetic nephropathy in NIDDM was significantly inhibited by good glycemic control (FPG < 140 mg/100 mL and HbA1 < 9.0%), independent of hypertension, and probably irrespective of the mode of therapeutic intervention.

Epistasis, photoreactivation and mutagen sensitivity of DNA repair mutants upr-1 and mus-26 in Neurospora crassa.

Double mutants were constructed combining mus-26, formerly designated uvs-(SA3B), with other UV-sensitive mutants. Tests of sensitivity of these double mutants to UV and to chemical mutagens revealed that mus-26 and upr-1 belong to the same epistatic group. The UV dose-response curve of mus-26 showed a characteristic plateau in the range of 100-200 J/m2. The same characteristic was also shown in the dose-response curves of upr-1 and the double mutant, upr-1 mus-26. Photoreactivation of UV damage in mus-26, upr-1 and upr-1 mus-26 was defective but not null. Assays were made of the reversion rate of ad-8 in strains that also carried UV-sensitive mutations. The reversion frequencies of the strains with upr-1 and upr-1 mus-26 were very low for the UV dose range below 300 J/m2, similarly to mus-26. Previously reported homozygous sterility of mus-26 was not caused by the mus-26 locus itself, and fertile strains were obtained among progeny. The results of this study suggest that mus-26 and upr-1 have similar properties in DNA repair.

Mutagenesis and epistatic grouping of the Neurospora meiotic mutants, mei-2 and mei-3, which are sensitive to mutagens.

To understand the possible roles of the Neurospora meiotic genes mei-2 and mei-3 in DNA repair, the frequencies of spontaneous and UV-induced mutation at the ad-3 loci were investigated and double mutants between mei mutants and other DNA-repair mutants were analyzed for mutagen sensitivity. Spontaneous mutation frequency in mei-2 was similar to that of the wild type, while the frequencies were high in both of two mei-3 strains which contained different mei-3 alleles. In addition, the frequency of spontaneous mutation in mei-3 varied greatly from experiment to experiment, which clearly showed a mutator phenotype for mei-3 mutation. UV irradiation increased mutation frequencies in both mei-2 and mei-3. The mutagen sensitivity of the double mutant, mei-2 mei-3, was no greater than that of the single mutants when treated with UV and methyl methanesulfonate (MMS). Thus, both mei genes appear to be involved in the same repair group. When analyzed in combination with other mutations, both mei mutations showed an epistatic relationship to uvs-6 and a synergistic relationship to mus-18 and uvs-2. However, uvs-3 was epistatic to mei-2, but the relationship of uvs-3 to mei-3 could not be tested directly, because the double mutant was barely viable. These results indicate that mei-2 and mei-3 are involved in the same repair group as uvs-6, and uvs-3 is possibly involved in the same group. Alternatively, the mei genes, or uvs-3 itself, may be related to more than one DNA repair group.

Isolation and characterization of MMS-sensitive mutants of Neurospora crassa.

Seven different mutants that show high sensitivity to MMS killing were isolated and mapped at different loci. One group, mms-(SA1), mms-(SA2) and mms-(SA6), showed high sensitivity to MMS but not to UV or gamma-rays. Another group, mms-(SA4) and mms-(SA5), showed extremely high sensitivity to UV and MMS. And mms-(SA3) and mms-(SA7) were moderately sensitive to both UV and MMS. Mms-(SA4) and mms-(SA1) were identified as alleles of uvs-2 and mus-7, respectively, which had been previously isolated. The mms-(SA1), mms-(SA6) and mms-(SA7) strains were barren in homozygous crosses, and the mms-(SA5) strain was barren in heterozygous crosses. The mms-(SA1), mms-(SA3) and mms-(SA5) strains showed high sensitivity to histidine. In summary, at least two new loci involved in the repair of MMS damage have been identified. The possibility that some of these new mutants are in new repair pathways is suggested.

A new ultraviolet-light sensitive mutant of Neurospora crassa with unusual photoreactivation property.

A mutant, uvs-(SA3B), which shows high sensitivity to UV light segregated among the progeny in a back-cross of a presumptive MMS-sensitive mutant to a wild-type strain. At 37% survival, this mutant was approximately 5 times more sensitive to UV and also 6 times more sensitive to 4-NQO than the wild type. But it was only slightly sensitive to gamma-ray, MMS, MNNG, MTC and histidine. It showed an unusual photoreactivation response. Its time course of photorecovery was similar to the photoreactivation-defective strain upr-1 of Neurospora crassa. Mutation induction by UV at the ad-3 loci in this mutant strain was lower than that at the same loci in the wild-type strain. The uvs-(SA3B) mutant maps between met-1 and col-4 in linkage group IV, and it was not allelic with the mutagen-sensitive mutant mus-8 which is located in this area. We have concluded, therefore, that uvs-(SA3B) has resulted from mutation in a new DNA-repair gene. This new mutant was barren in homozygous crosses.

Effects of heat shock on the induction of mutations by chemical mutagens in Neurospora crassa.

Preheating of Neurospora conidia increased their susceptibility to mutation induction by chemical mutagens. Optimal conditions of heat shock for enhanced mutagenesis were determined in 2.5 X 10(7) conidia/ml 0.067 M KH2PO4-Na2HPO4 (pH 7.0) buffer to be treatment at 43 degrees C for 60 min. When protein synthesis during heat stock was eliminated by cycloheximide or by use of the temperature-sensitive mutation psi-1, induction of thermotolerance was inhibited while induction of the enhanced state of mutability was not. Therefore, inducible protein synthesis is not involved in this process. To discover whether DNA-repair systems are altered by heat shock and, as a result, whether reversion frequencies increase, DNA-repair mutants (upr-1, uvs-2, uvs-3, uvs-6, mus-7, mus-16) were heated and their reversion frequencies at the ad-8 locus were measured. All the DNA-repair mutants showed higher reversion frequencies with MNNG treatment after heat shock than in non-heated control. It therefore seems that DNA repair is not involved in the enhancement of chemical mutagenesis by heat shock. Heat shock does not increase frequencies of reversion induced by ultraviolet light, and heat shock after treatment with chemical mutagens does not affect reversion frequencies. These results suggest that heat shock may change the structure and function of cellular membranes and thereby increase the influx of mutagens into cells.