Clinical features and skewed X-chromosome inactivation in female carriers of X-linked recessive spinal and bulbar muscular atrophy.

In X-linked recessive disorders, a few female gene carriers become symptomatic. Recent evidence implicates skewed X-chromosome inactivation in such female carriers. We studied the clinical features of eight female gene carriers of X-linked recessive spinal and bulbar muscular atrophy (SBMA), and evaluated the relationship between phenotype and genotype from the viewpoint of X-chromosome inactivation. Seven of eight cases were symptomatic, showing mild muscle weakness, frequent muscle cramps, slight elevation of the serum creatinine kinase level, or neurogenic changes on the electromyogram. Only one carrier was asymptomatic clinically. For the estimation of X-chromosome inactivation, the methylation status of the androgen receptor (AR) gene was determined by polymerase chain reaction-based assay. Highly skewed inactivation of the affected AR gene was found in the asymptomatic carrier, while symptomatic carriers had a random or lower inactivation pattern of the affected AR gene. These findings suggest that most female carriers of SBMA show some clinical abnormalities, and highly skewed inactivation of the affected X-chromosome seems to closely relate with escape of the manifestation in female carriers of SBMA.

[Relationship between changes in skin lipids by ultraviolet exposure and the cell toxicity].

The oxidative decomposition of skin lipids by UV exposure and its cell toxicity were studied in vivo and in vitro, using guinea pigs, to investigate the role of oxidative damage in cell membranes and mitochondria in nuclear genome DNA damage resulting in skin cell death by ultraviolet (UV) exposure. Two new methods were developed for this research: selective methylation by trimethylsilyldiazomethane (TMSCHN2) of free fatty acids in crude skin lipids for gas chromatography-mass spectrometry (GC-MS) analysis, and the improvement of the thiobarbituric acid (TBA) reaction for lipid peroxide analysis, which was obtained by adding 0.01% BHT and 1 mM EDTA, an antioxidant additive, into the reaction system described by Ohkawa. The following findings were noted: 1. Using an optical microscope, the infiltration of inflammatory cells such as neutrophils, lymphocytes and monocytes was noted after skin tissue was exposed to UV. 2. The increase of lipid peroxide in exposed areas was small. In the epidermis the increase was about 2 times higher than the non-exposed areas. 3. The fragments of lipid were generated in accordance with the increase of free fatty acids (C16:0, C18:1,:2, C18:0) in the surface of the exposed skin by GC-MS precise assay, and the level of 7-dehydrocholesterol was decreased. 4. The skin homogenate received peroxidation by ultraviolet more easily than living skin, and its peroxidation was inhibited with fat-soluble antioxidative agents such as flavonoids, BHT, BHA, and vitamin E and the metal chelating agent such as Fenton reaction inhibitor as expected; however, it was promoted by water soluble antioxidative agents such as glutathione and vitamin C, which are useful to the human body. 5. Steroids and nonsteroidal anti-inflammatory drugs (NSAIDs) such as indomethacin and aspirin, had no inhibitory effect on lipid peroxidation by ultraviolet as properties of chemicals.

Influence of initial status on functional gain for Japanese patients with first cerebral hemorrhage.

It is important to identify in advance patients who will achieve the greatest functional gains from rehabilitation therapy, as specialist rehabilitation resources are still scarce in Japan. The purpose of this study was to determine whether functional score at admission influences the functional change (functional score at discharge minus functional score at admission) after inpatient rehabilitation for first cerebral hemorrhage. One hundred and ninety-three patients with cerebral hemorrhage were enrolled in this study. They were assessed using the Functional Independence Measure (FIM) at admission and discharge and underwent inpatient rehabilitation treatment. Patients were stratified into 3 groups according to their FIM total scores on admission as follows: (1) < or = 36 (severely affected patient group); (2) 37-72 (moderately affected patient group); and (3) >73 (mildly affected patient group). Scheffe's multiple comparison test showed that patients in group 1 were significantly older (mean +/- SD = 63 +/- 10 years) than those in groups 2 (56 +/- 10 years) or 3 (53 +/- 12 years). Patients in group 2 showed significantly greater FIM gain (37 +/- 17) compared with patients in groups 3 (23 +/- 12) or 1 (27 +/- 23). The results suggest that moderately affected patients at admission will show significantly higher functional gain compared with severely or mildly affected patients. Mildly affected patients at admission had a significantly shorter length of hospital stay for rehabilitation than the other groups. There was no significant difference in onset to admission interval between the 3 groups. The functional levels of affected patients on admission, as stratified by the FIM scale, roughly predict the degree of functional gain following rehabilitation in patients with first cerebral hemorrhage. Moderately affected patients will benefit from intensive rehabilitation. This study may be useful in determining how best to prioritize rehabilitation therapy.

Influence of admission functional status on functional change after stroke rehabilitation.

OBJECTIVE: To determine whether the admission functional score influences the functional change after stroke rehabilitation. DESIGN: Two hundred forty-three patients who had received the Functional Independence Measure (FIM) assessment at admission and at discharge were enrolled in the study. The patients were stratified into three groups according to their FIM total scores at admission, i.e., < or =36, 37 to 72, and > or =73. RESULTS: The Scheffé's multiple comparison test showed that patients with FIM total scores of > or =73 at admission were significantly younger (58 +/- 11 [SD] yr) than those who had scores of 37 to 72 (64 +/- 11 yr) or < or =36 (66 +/- 12 yr). Patients with FIM total scores of 37 to 72 at admission showed significantly higher FIM gain (37 +/- 15) compared with those patients who had scores of > or =73 (20 +/- 10) or < or =36 (29 +/- 23). CONCLUSION: The functional levels of affected patients at admission stratified by the FIM scale roughly predict the degree of functional gain after rehabilitation in survivors with a first episode of ischemic stroke. Moderately affected patients will benefit from intensive rehabilitation. These findings may be useful for rehabilitation triage.

Methylenetetrahydrofolate reductase gene polymorphism and ischemic stroke: sex difference in Japanese.

Moderately elevated plasma homocysteine levels have been established as independent risk factors in vascular disease, including ischemic stroke. Recently, a common mutation (C677T) in the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene reducing the activity of MTHFR and increasing homocysteine levels in plasma was reported. The C677T MTHFR mutation may be a risk factor for ischemic stroke, but the results of previous studies have been conflicting. One possible explanation is that the association with the MTHFR genotype may be different according to gender. To investigate the association for ischemic stroke, we conducted a case-control study of 77 hospital cases (49 men and 28 women) with ischemic stroke and 229 (120 men and 109 women) control subjects in Japanese. The prevalence of conventional vascular risk factors and MTHFR genotypes were determined in case and controls. After adjustment by multiple analysis in all there was no statistical significance in MTHFR genotypes. The conventional vascular risk factors such as diabetes mellitus (adjusted odds ratio [OR], 17.21), hypertension (adjusted OR, 4.67), smoking habit (adjusted OR, 4.70), and hyperlipidemia (adjusted OR, 2.73) were identified independently associated with ischemic stroke. With a separate sex analysis it was identified that the relationship of the MTHFR T/T gneotype was statisticaly significant in women (adjusted OR, 9.49; 95% CI, 1.75-51.47, P=0.0091). The relevance of the MTHFR T/T mutation appears to be restricted to women, suggesting a role of female hormones in the resistance to elevated homocysteine levels due to the MTHFR T/T mutation.

Comparative analysis of plasma and erythrocyte 7-ketocholesterol as a marker for oxidative stress in patients with diabetes mellitus.

OBJECTIVES: To reveal increased lipid peroxidation in diabetics by quantification of cholesterol oxidation products (COPs) not only in plasma, but also in erythrocytes. DESIGN AND METHODS: We quantified 7-ketocholesterol (7-kCho) by gas chromatography-mass spectrometry as a surrogate measure for COPs. These assays were performed on both plasma and erythrocytes in 20 control subjects and 20 treated patients with relatively poorly controlled Type 2 diabetes. RESULTS: Both plasma and erythrocyte 7-kCho levels in diabetics were significantly higher than those in control subjects. Although neither plasma nor erythrocyte 7-kCho levels were associated with markers for glucose tolerance in diabetics, a negative correlation of serum HDL-cholesterol levels with erythrocyte, but not plasma, 7-kCho levels was found. CONCLUSION: Increased oxidative stress in diabetics affects oxidation of cholesterol. Assays of COPs not only in plasma, but also in erythrocytes, may yield complementary information in lipid peroxidation.

Identification, quantitative determination, and antioxidative activities of chlorogenic acid isomers in prune (Prunus domestica L. ).

Neochlorogenic acid (3-CQA) and cryptochlorogenic acid (4-CQA), isolated from prune (Prunus domestica L.), were identified by NMR and MS analyses. In addition, the quantity of chlorogenic acid isomers in prune were measured by HPLC. These isomers, 3-CQA, 4-CQA, and chlorogenic acid (5-CQA), were contained in the ratio 78.7:18. 4:3.9, respectively. 4-CQA was identified and quantified in prune for the first time, and relatively high amounts of this isomer were characteristic. Antioxidative activities of the chlorogenic acid isomers, such as scavenging activity on superoxide anion radicals and inhibitory effect against oxidation of methyl linoleate, were also evaluated. Each isomer showed antioxidative activities which were almost the same.

The C677T mutation in the methylene tetrahydrofolate reductase gene increases serum uric acid in elderly men.

A common mutation, C677T, in the methylene tetrahydrofolate reductase gene (MTHFR) reduces the activity of MTHFR and increases total homocysteine levels in plasma. Increased homocysteine levels are reportedly associated with high serum uric acid levels. The relationship between the MTHFR mutation and uric acid metabolism remains unclear, however. To investigate whether the C677T MTHFR mutation is a risk factor for hyperuricemia, we performed MTHFR genotyping and clinical laboratory determinations, including serum uric acid, in 271 elderly Japanese men (age range, 40-79 years; mean, 52.6 years). The mean uric acid levels for the C/C, C/T, and T/T genotypes were 5.67, 6.00, and 6.39 mg/dl, respectively (P = 0.012). The T/T genotype was more frequent in subjects with high uric acid levels than in those with low uric acid levels (P = 0.038). These findings suggest that the C677T MTHFR mutation contributed to higher uric acid levels in subjects enrolled in this study. In conclusion, the mutation of the MTHFR gene may be a risk factor for hyperuricemia in elderly men.

Nilvadipine protects low-density lipoprotein cholesterol from in vivo oxidation in hypertensive patients with risk factors for atherosclerosis.

OBJECTIVE: Nilvadipine, a calcium antagonist, has been shown to have antioxidant activity in vitro, but its effect on in vivo oxidation has not been assessed. The aim of this study was to investigate the antioxidant effect of this agent in vivo. The ratios of 7-keto cholestadien to cholesterol are believed to be an available marker of lipid peroxidation. Using these ratios, we have assessed the antioxidant effect of nilvadipine on low-density lipoprotein (LDL) in hypertensive patients with high risk of atherosclerosis. The risk factors of atherosclerosis may involve oxidation of LDL. METHODS: Fifteen healthy subjects (seven females and eight males aged 35-72 years, mean +/- SD = 55.3 +/-13.8 years) and fifteen hypertensive patients (seven females and eight males aged 45-80 years, mean +/- SD = 60.2 +/- 11.8 years) were recruited. Patients were treated orally with nilvadipine (4 mg b.i.d.) for 4 weeks. Cholesterol oxidation levels of LDL in patients before and after nilvadipine therapy and healthy subjects were studied. RESULTS: The ratios of 7-keto cholestadien to cholesterol in LDL of hypertensive patients before and 4 weeks after nilvadipine treatment and in healthy subjects were 6.5 +/- 1.6% (mean +/- SD), 3.8 +/- 1.2%, and 0.2 +/- 0.1%, respectively. There were significantly (P < 0.001) increased levels of cholesterol oxidation in LDL in patients with hypertension both before and after nilvadipine treatment compared with healthy subjects. However, there was a significantly (P < 0.001) decreased level of cholesterol oxidation in LDL in patients after nilvadipine treatment compared with patients before nilvadipine treatment. CONCLUSION: Our data showed that nilvadipine may protect LDL cholesterol from in vivo oxidation in hypertensive patients with high risk of atherosclerosis.

Analysis of 7-ketocholesterol in low density lipoprotein and fatty acid composition in erythrocyte membranes of patients on maintenance hemodialysis and healthy controls.

We established a method to quantify 7-ketocholesterol (7-KC) in low density lipoprotein by using the heparin-citrate method and gas chromatography-mass spectrometry. We examined the concentration of 7-ketocholesterol in LDL using this method to assess the pathological conditions in uremic patients with hemodialysis and healthy controls. We also examined the fatty acid composition in erythrocyte membranes to estimate the modification of biological membranes. We showed that the concentrations of 7-KC/cholesterol in LDL were significantly increased in hemodialysis patients compared to healthy controls (3.68+/-0.45 vs. 2.41+/-0.19, P<0.05) and the ratio of polyunsaturated fatty acids to saturated fatty acids in erythrocyte membranes was significantly decreased in hemodialysis patients compared to healthy controls (0.499+/-0.014 vs. 0.655+/-0. 017, P<0.001). There was no significant difference in 7-KC concentration in LDL or fatty acid composition in erythrocyte membranes between pre- and post-intervention of hemodialysis. We concluded that hemodialysis patients are under oxidative stress, which modifies LDL and erythrocyte membranes and we speculated these modifications may participate in the process of atherosclerosis. We believe that the method to quantify 7-KC in this study is concise and reliable and may be used to investigate various diseases.

The timing of GM-CSF expression plasmid administration influences the Th1/Th2 response induced by an HIV-1-specific DNA vaccine.

The mechanism of immune activation induced by a plasmid-encoding GM-CSF (pGM-CSF), administered in combination with a DNA vaccine encoding the envelope of HIV, was studied. Injecting pGM-CSF i.m. into mice 3 days before DNA vaccination primarily induced a Th2 response. Simultaneous administration of the DNA vaccine plus pGM-CSF activated both a Th1 and a Th2 response. When the plasmid was injected 3 days after DNA vaccination, enhancement of Th1 immunity predominated. These results suggest that the timing of cytokine expression determines the phenotype of the resultant Th response. After 3 days of pGM-CSF injection, the increased percentages of CD11c+, CD8+ cells were observed in the regional lymph nodes. In addition, many infiltrated cells, including S-100 protein-positive cells, were found in the pGM-CSF-injected tissue. The importance of these S-100+ cells or both CD8+ and CD11c+ cells, especially that of dendritic cells (DCs), was also studied. DCs derived from bone marrow and cultured in RPMI 1640 medium containing IL-4 and GM-CSF were incubated with DNA vaccine and then transferred into naive mice. Mice receiving DCs showed strong HIV-1-specific Th2 immune responses. Our results suggest that DCs play important roles in the activation or modification of the Th2-type immune response induced by DNA vaccination.

Molecular genetic analysis and mutation screening of the VHL gene in a Japanese family with von Hippel-Lindau disease.

Von Hippel-Lindau (VHL) disease is an autosomal dominantly inherited familial cancer syndrome which predisposes individuals to a variety of malignant and benign tumors. Its penetrance is almost 100% by 61-70 years of age. We have identified a germline mutation in the VHL gene of a Japanese male patient with a retinal hemangioma in the left eye, an intracranial hemangioblastoma, a left renal tumor and bilateral pheochromocytomas. Screening for the mutation was performed in all members of the patient's family at risk for VHL disease. The mutation identified in the patient was a missense mutation at codon 238 (CGG to CAG). The elder daughter, who did not show any clinical symptoms, was found to carry the same mutation. In order to detect and treat tumors in VHL patients at an earlier stage, it is necessary to identify and screen for the VHL gene mutation in all family members known at risk.

Dicarboxylic acids as markers of fatty acid peroxidation in diabetes.

Increased urinary excretion of dicarboxylic acids (DAs) has been well known in patients with diabetic ketoacidosis (DKA). It was known that small amounts of such DAs were also detected in urine from healthy humans. Upon chemical, radiation-induced or enzymatic oxidation, cis-polyunsaturated fatty acids (PUFA) have previously been shown to generate saturated short- and medium-chain length DAs. In diabetes, it was confirmed that the imbalance between the generation of free radicals and antioxidant defense systems increases oxidative stress and leads to the damage of lipid, which contains PUFA. Some peroxidation products of PUFA, such as malondialdehyde and conjugated diene, are generally known to be elevated in patients with diabetes. The present study was undertaken to determine if urinary excretion of DAs is elevated in diabetic patients without DKA. Urine samples from ten non-ketoacidotic patients with type 2 diabetes and ten healthy subjects were examined for DAs by combined gas chromatography and mass spectrometry with selected ion monitoring. The diabetic subjects had significantly (Psebacic acid. Being stable and easily detectable compounds, DAs may be considered potential markers of oxidative attack on PUFA in diabetes.

Detection of mutations in the COL4A5 gene in over 90% of male patients with X-linked Alport's syndrome by RT-PCR and direct sequencing.

X-linked Alport's syndrome is caused by mutations in the COL4A5 gene encoding the type IV collagen alpha5 chain (alpha5[IV]). Polymerase chain reaction-single-str and conformation polymorphism (PCR-SSCP) on genomic DNA has previously been used to screen for mutations in the COL4A5 gene, but this method was relatively insensitive, with mutations detected in less than 50% of patients. Here, we report a systematic analysis of the entire coding region of the COL4A5 gene, using nested reverse-transcription-polymerase chain reaction (RT-PCR) and the direct sequence method using leukocytes. This study examines twenty-two unrelated Japanese patients with X-linked Alport's syndrome showing abnormal expression of alpha5(IV) in the glomerular or epidermal basement membranes. Mutations that were predicted to be pathogenic were identified in 12 of the 13 male patients (92%) and five of the nine female patients (56%). Six patients had missense mutations, four had out-of-frame deletion mutations, three had nonsense mutations, and three had mutations causing exon loss of the transcript. The current study shows that nested RT-PCR and the direct sequence method using leukocytes are highly sensitive and offer a useful approach for systematic gene analysis in patients with X-linked Alport's syndrome.

Hybrid survival motor neuron genes in Japanese patients with spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a frequently occurring autosomal recessive disease, characterized by the degeneration of spinal cord anterior horn cells, leading to muscular atrophy. Most SMA patients carry homozygous deletions of the telomeric survival motor neuron gene (SMN) exons 7 and 8. In the study presented here, we examined 20 Japanese SMA patients and found that 4 of these patients were lacking in telomeric SMN exon 7, but retained exon 8. In these 4 patients, who exhibited all grades of disease severity, direct sequencing analysis demonstrated the presence of a hybrid SMN gene in which centromeric SMN exon 7 was adjacent to telomeric SMN exon 8. In an SMA family, a combination of polymerase chain reaction and enzyme-digestion analysis and haplotype analysis with the polymorphic multicopy marker Agl-CA indicated that the patient inherited the hybrid gene from her father. In conclusion, hybrid SMN genes can be present in all grades of disease severity and inherited from generation to generation in an SMA family.

Link between glycation and lipoxidation in red blood cells in diabetes.

Oxidative stress is postulated to be increased in patients with diabetes mellitus. Glycation enhanced by elevated glucose concentrations may induce the formation of oxygen-derived free radicals (OFRs). OFRs would cause oxidative damage to endogenous molecules, including cholesterol. Accumulating evidence suggests that oxidative cell injury caused by OFRs contributes to the development of both macroangiopathy and microangiopathy in diabetes. Our previous studies have shown that 7-keto cholestadien is one of the major products of cholesterol peroxidation in diabetic erythrocyte membrane and its levels correlate with hemoglobin Alc (HbAlc) values. We have newly identified 3-cholesten-6-one, one of the minor products of cholesterol peroxidation, in it. The aim of our study is to investigate whether 3-cholesten-6-one levels also correlate with HbAlc values. Levels of 3-cholesten-6-one were assessed in erythrocyte membrane lipid by monitoring peak areas of 3-cholesten-6-one to cholesterol with gas chromatography-mass spectrometry. The peak area ratio of 3-cholesten-6-one to cholesterol was used as a marker of cholesterol peroxidation. The HbAlc value, an index of both glycemic stress and glycation, was measured by high-performance liquid chromatography. In this study, we evaluated 33 diabetic and 29 healthy subjects, matched for age (59.3+/-14.5 vs. 57.3+/-13.7 years, mean+/-S.D.) and sex (15 males and 14 females vs. 16 males and 17 females). There were both significantly raised HbAlc levels (4.6+/-0.8 vs. 8.3+/-2.4%, P<0.001) and significantly increased ratios of 3-cholesten-6-one to cholesterol (0.2+/-0.4 vs. 21+/-1.8, P<0.001) in diabetic patients compared to control subjects. A good correlation between HbAlc levels and ratios of 3-cholesten-6-one to cholesterol was found in participants (r = 0.75, P<0.001, y = 0.46x-1.8). This suggests that an oxidative stress exists in diabetes and the link between glycation and lipoxidation is found in diabetic red blood cell.

Formation of 9-hydroxy linoleic acid as a product of phospholipid peroxidation in diabetic erythrocyte membranes.

The increased production of oxygen-derived free radicals (OFR) and lipid peroxidation may contribute to vascular complications in diabetes. Some lipid peroxidation products have already been reported to be formed via glucose-induced oxidative stress. We have identified 9-hydroxy linoleic acid (9-OH-C18:2) in the red cell membrane phospholipid of diabetic subjects. We hypothesized that 9-OH-C18:2 would be formed in hydroxyl radical reactions to linoleic acid (C18:2) during glucose-induced oxidative stress, and confirmed that the formation of 9-OH-C18:2 was induced by ultraviolet (UV)-C irradiation to the synthetic C18:2. UV-C light generates highly reactive hydroxy radicals. C18:2 is confirmed to be the precursor of 9-OH-C18:2. To estimate the degree of oxidative damage to red cell membrane phospholipids, we developed a selective ion monitoring gas chromatography-mass spectrometric measurement for C18:2 and 9-OH-C18:2, following methanolysis of red cell membrane phospholipids. The relative peak height ratio of C18:2 to 9-OH-C18:2 (9-OH-C18:2/C18:2) was measured in phospholipid extracts of red cell membranes from healthy (n=29, 3.1+/-1.9%) and diabetic (n=27, 20. 9+/-16.1%) subjects. It was confirmed that 9-OH-C18:2/C18:2 is significantly (P<0.001) elevated in patients with diabetes. The measurement of 9-OH-C18:2/C18:2 in red cell membranes should be useful for assessing oxidative damage to membrane phospholipids in diabetes.

High incidence of a survival motor neuron gene/cBCD541 gene ratio of 2 in Japanese parents of spinal muscular atrophy patients: a characteristic background of spinal muscular atrophy in Japan?

Most spinal muscular atrophy (SMA) patients lack the survival motor neuron gene (SMN). However, the patients retain at least one copy of the cBCD541 gene (BCD), which is highly homologous with SMN. Here, we determined the SMN/BCD copy number ratios (the S/B ratios) of 12 parents of Japanese SMA patients with a homozygous SMN deletion, using competitive oligonucleotide priming polymerase chain reaction. We identified an S/B ratio of 2 in 25% of the parents examined, whereas less than 2% of parents of SMA patients in Western populations have an S/B ratio of 2. The high incidence of an S/B ratio of 2 in Japanese parents of SMA patients may reflect the characteristic genetic background of SMA in Japan.

Glycated hemoglobin and lipid peroxidation in erythrocytes of diabetic patients.

In diabetes, glycation and subsequent browning (or glycoxidation) reactions are enhanced by elevated glucose concentrations. It is unclear whether the diabetic state per se also induces an increase in the generation of oxygen-derived free radicals (OFRs). However, there is some evidence that glycation itself may induce the formation of OFRs. OFRs cause oxidative damage to endogenous molecules, including cholesterol. 7-Oxocholesterol is known to be one of the major products of cholesterol oxidation. The level of cholesterol peroxidation products was assessed in erythrocyte membrane lipid by monitoring the peak height ratio of 7-oxocholesterol, one of the products of cholesterol peroxidation, to cholesterol with gas chromatography/mass spectrometry (GC/MS). The peak height ratio of 7-oxocholesterol to cholesterol was used as a biomarker of lipid peroxidation. The hemoglobin A1c (HbA1c) value, an index of glycemic stress, was measured by high-performance liquid chromatography. We examined the relationship between the levels of cholesterol peroxidation products and HbA1c in erythrocytes of diabetic and healthy subjects. There was a significantly increased ratio of 7-oxocholesterol to cholesterol in diabetic erythrocytes compared with control erythrocytes. The ratio of 7-oxocholesterol to cholesterol was significantly correlated with the level of HbA1c. This suggests that glycation of hemoglobin via chronic hyperglycemia is linked to cholesterol peroxidation in erythrocytes of both diabetic and healthy subjects.