Human CYP1A2: sequence, gene structure, comparison with the mouse and rat orthologous gene, and differences in liver 1A2 mRNA expression.

We have sequenced the human CYP1A2 (cytochrome P(3)450) gene, 1,906 basepairs (bp) of the 5' flanking region, and 113 bp of the 3' flanking region. The gene spans almost 7.8 kilobases, comprising seven exons and six introns. The transcriptional start site was determined by both primer extension and S1 mapping. Including the first noncoding exon of 55 bp, the entire mRNA is 3,121 bp in length, and the open reading frame, starting with nucleotide 10 of exon 2, encodes 515 amino acids (mol wt = 58,294). Between the human CYP1A2 and CYP1A1 (cytochrome P(1)450) genes, exons 2, 4, 6, and especially 5 are strikingly conserved in both nucleotide similarity and total number of bases. Alignment of the upstream sequences and exon 1 of human CYP1A2 with that of mouse or rat CYP1A2 revealed two possibly significant regions of similarity: 1) 68% in the approximately 150 bases immediately 5' from the mRNA cap site and 2) 80% identify between the human -841 to -758 segment and the mouse -1,529 to -1,439 segment. The canonical 5-bp box (CACGC), found upstream of all mammalian CYP1A1 genes to date and believed to interact with the inducer.aromatic hydrocarbon receptor complex, was not found on either strand in the 1,906 bp of the 5' flanking region of human CYP1A2. In contrast, alignment of the upstream sequences, exon 1, and intron 1 of human CYP1A1 with that of mouse or rat CYP1A1 revealed large, highly conserved regions. Conserved regions were found in intron 1 of the human, mouse, and rat CYP1A2 gene. These data suggest that the regulatory elements controlling the CYP1A2 gene might differ in location from those controlling the CYP1A1 gene. Among 12 human liver samples, striking differences (greater than 15-fold) in the 3.3-kilobase 1A2 mRNA levels were seen. This result may reflect significant genetic differences in constitutive and/or inducible CYP1A2 gene expression that could play an important role in individual risk of environmental toxicity or cancer.

Somatic mosaicism for a DMD gene deletion.

Mosaicism is a mixed state, with two cell populations of different genetic origins caused by a cell mutation occurring after fertilization. In the present case, DNA analysis of lymphocytes led to a DMD diagnosis before death. Postmortem immunocytochemical and DNA analysis showed somatic mosaicism. At age 18 years, blood lymphocyte DNA analysis showed a DMD gene deletion, upstream from exon 7 to the 5' end containing both muscle and brain promoters. As the patient's mother and elder sister had no deletions, he was considered to have a new mutation. Immunocytochemical studies of postmortem tissues showed that dystrophin was absent from the tongue, deltoid, intercostal, psoas and rectus femoris muscles, but there was a mix of dystrophin-positive and negative fibers in the rectus abdominis, cardiac, temporalis and sternocleidomastoid muscles. All diaphragm cells were dystrophin positive. Polymerase chain reaction (PCR) amplification from all tissues except the temporalis and sternocleidomastoid muscles, diaphragm and kidney, in which no deletion was found, showed the deletion from at least exon 6 to the 5' end containing both muscle and brain promoters. In this case, a genomic deletion of the DMD gene contributed to the formation of tissues derived from both ectoderm and endoderm, and cells of mesodermal origin showed genotypic and phenotypic heterogeneity. Our results indicate a mutation of the present case may have occurred just before the period of germ layer formation.

Molecular genetic and immunological analysis of dystrophin of a young patient with X-linked muscular dystrophy.

We examined the nucleotide sequence of deleted part of dystrophin mRNA and its translational product with immunoblot and immunohistochemical methods in a 6-year-old boy with a deleted DMD/BMD gene. On Southern blot analysis of his genomic DNA, we found a deletion of exons 10 to 37 in the DMD/BMD gene, which was expected to preserve the translational open reading frame (ORF). Dystrophin mRNA from his biopsy sample was amplified by polymerase chain reaction (PCR) and sequenced. The mRNA lacked the sequence corresponding to the gene from exons 10-37, and the translational ORF was preserved. The transcript was expected to code a 260 kDa protein. Dystrophin expressed in this patient was investigated with immunological methods. A 260 kDa protein was detected by immunoblot analysis with antidystrophin antiserum against nondeleted regions. These observations confirmed the preservation of the reading frame and the 260 kDa protein was produced as a mutant dystrophin. All these are compatible with the diagnosis of BMD. However, the immunohistochemical pattern of his muscle cells was peculiar. With deleted-region-directed antiserum, the membrane was not stained at all as in DMD patients. In contrast, with nondeleted-region-directed antiserum, all the muscle cell membrane was stained continuously as in non-DMD/BMD individuals. These are quite different from the staining pattern in most BMD patients where muscles are stained patchily or discontinuously.

Prenatal diagnosis of Fukuyama type congenital muscular dystrophy in eight Japanese families by haplotype analysis using new markers closest to the gene.

We conducted prenatal diagnosis by haplotype analysis, using newly developed microsatellite markers, in eight Fukuyama type congenital muscular dystrophy (FCMD) families. In addition to six new families, two previously reported families were reexamined by haplotype analysis including detection of an ancestral founder haplotype (138-183-301) for 3 microsatellite markers closest to the FCMD gene, designated D9S2105-D9S2107-D9S172, the distances of which from the FCMD gene are presumed to be approximately 140, approximately 20, and approximately 280 kb, respectively. Five fetuses from five families were diagnosed as nonaffected, and were subsequently confirmed to be healthy. Three fetuses of the other three families were diagnosed as having a high probability of being affected by FCMD. In the prenatal diagnosis conducted for these eight families, the ancestral founder allele was observed in 13 of 16 (81%) FCMD-bearing chromosomes. Detection of the ancestral haplotype facilitated achieving accurate prenatal diagnosis of FCMD. The brains of all three fetuses prenatally diagnosed as FCMD-affected showed the initial stage of cortical dysplasia, strong evidence of FCMD.

Haplotype-phenotype correlation in Fukuyama congenital muscular dystrophy.

In typical Fukuyama congenital muscular dystrophy (FCMD), peak motor function is usually only unassisted sitting or sliding on the buttocks, though a few patients are able to walk at some point. However, a few patients have a severe phenotype and never acquire head control. In addition, it is clinically difficult to differentiate this severe FCMD from Walker-Warburg syndrome (WWS) or from muscle-eye-brain disease (MEBD). In order to establish a genotype-phenotype correlation, we performed haplotype analysis using microsatellite markers closest to the FCMD gene (FCMD) in 56 Japanese FCMD families, including 35 families whose children were diagnosed as FCMD with the typical phenotype, 12 families with a mild phenotype, and 9 families with a severe phenotype. Of the 12 propositi with the mild phenotype, 8 could walk and the other 4 could stand with support; 10 cases were homozygous for the ancestral founder (A-F) haplotype whereas the other 2 were heterozygous for the haplotype. In the 9 severe cases, who had never acquired head control or the ability to sit without support, 3 had progressive hydrocephalus, 2 required a shunt operation, and 7 had ophthalmological abnormalities. Haplotype analysis showed that 8 of the 9 cases of the severe phenotype are heterozygous for the A-F haplotype, and the other one homozygous for the haplotype. We confirmed that at least one chromosome in each of the 56 FCMD patients has the A-F haplotype. The rate of heterozygosity for the A-F haplotypes was significantly higher in severe cases than in typical or mild cases (P < 0.005). Severe FCMD patients appeared to be compound heterozygotes for the founder mutation and another mutation. Thus, the present study yielded molecular genetic evidence of a broad clinical spectrum in FCMD.

[Effects of steroids on hepatic ATP and L/P ratio in rats subjected to acute hemorrhage].

Both steroids and hemorrhage may affect the hepatic energy metabolism. The effects of steroids (5 mg.kg-1 of methylpredonisolone, 50 mg.kg-1 of methylpredonisolone, 25 mg.kg-1 of hydrocortisone and 250 mg.kg-1 of hydrocortisone) on hepatic ATP level and L/P ratio were evaluated in rats under acute hemorrhage. There were no significant differences in the hepatic ATP levels and L/P ratio among 5 groups. However, the base excess in 3 steroid groups (50 mg.kg-1 of methylpredonisolone, 25 mg.kg-1 of hydrocortisone and 250 mg.kg-1 of hydrocortisone) was significantly higher than that in the control group. This result suggests that steroids may improve the metabolic acidosis during acute hemorrhage.

[Effects of hypertonic lactated Ringer's solution on hepatic ATP and L/P ratio in rats subjected to acute hemorrhage].

This study aimed to clarify the difference in the effects of hypertonic lactated Ringer's solution (HLS) on hepatic ATP level and L/P ratio during acute hemorrhage in rats. The hepatic ATP level in HLS 230 group was lower, and L/P ratio in HLS 300 group was higher than those in the control group. There was no significant difference in glycogen among 4 groups. However, pH and the base excess in HLS 230 and HLS 300 group were significantly higher than those in the C group. Heart rate in HLS 300 group was significantly lower than that in the C group. These results suggest that HLS may not be useful with regard to the hepatic energy metabolism, although it improves the metabolic acidosis during acute hemorrhage.

[The influence of N-nitro-L-arginine methyl ester (L-NAME) on hydroxyl free radical formation in the post-ischemic reperfused heart of anesthetized rats].

N-nitro-L-arginine methyl ester (L-NAME) has been reported to have protective action against hydroxyl free radicals. We have investigated whether L-NAME influences free radical formation in the post-ischemic reperfused heart of anesthetized rats. An isolated rat heart-lung preparation was used. Forty male Wistar rats were allocated into D (D-NAME 100 microMol.l-1), L (L-NAME 100 microMol.l-1), LH (L-NAME 100 microMol.l-1 and 1MAC halothane), LI (L-NAME 100 microMol.l-1 and 1MAC isoflurane), and LS (L-NAME 100 microMol.l-1 and 1MAC sevoflurane) groups. The heart was perfused initially at the cardiac output of 30 ml.min-1 and the atrial pressure of 70 mmHg. Drugs were administered into the reservor 7 min after the start of perfusion. Ten minutes after the start of perfusion, the heart was rendered globally ischemic for 10 min by reducing the preload and afterload to zero and then reperfused for 10 min. At the end of reperfusion, the heart was freeze-dried for 4 days. The perfusate blood was collected just before and after ischemia and at the end of reperfusion. The formation of hydroxyl radicals in the perfusate blood and heart was measured with high-performance liquid chromatography using salicylic acid. Hydroxyl radicals react with salicylic acid, yielding dihydroxybenzoic acid (DHBA). Before and after ischemia, there were no significant differences among the groups in cardiac output, systolic pressure, heart rate, and right atrial pressure. DHBAs in the heart of L, LH, LI, and LS groups were significantly lower than those of D group. However, there were no differences in the DHBA levels among 4 groups. The concentrations of DHBA in the perfusate blood after ischemia and reperfusion were significantly higher than those before ischemia in all groups. DHBAs in the perfusate blood after ischemia and reperfusion of L, LH, LI, and LS groups were significantly lower than those of D group. However, there were no differences in the DHBA levels among 4 groups administered L-NAME. This study indicates that L-NAME reduces hydroxyl free radical formation in the post-ischemic reperfused heart in anesthetized rats and volatile anesthetics do not influence the depressant effect of hydroxyl free radical formation by L-NAME.

[Effects of lactated Ringer solution and acetated Ringer solution on hepatic ATP and L/P ratio in rats subjected to acute hemorrhage].

This study aimed to clarify the difference in the effects of lactated Ringer solution (LR) and acetated Ringer solution (AR) on hepatic ATP level and L/P ratio during acute hemorrhage in rats. There were no significant differences in the hepatic ATP levels and L/P ratios among 3 groups. Glycogen in LR group was higher than that in the control group. However pH and the base excess in LR and AR group were significantly higher than those in the C group. These results suggest that LR as well as AR may improve the metabolic acidosis, and LR may be more useful than AR with regard to glucose supply during acute hemorrhage.

[Anesthetic management of laparoscopic ovarian cystectomy in a 2-year-old child].

We have experienced the anesthetic management of laparoscopic ovarian cystectomy in a 2-year-old child. The patient was anesthetized with isoflurane and manually ventilated to keep PaCO2 at 30 mmHg prior to the carbon dioxide insufflation of the peritoneal cavity. Two minutes after the abdomen was distended, end-tidal CO2 began to increase and after 10 minutes PaCO2 was 50 mmHg. We had to increase tidal volume and respiratory rate, although airway pressure was 30 cmH2O. We should be careful because children who undergo pneumoperitoneum become hypercapnic easily during general anesthesia.

[Genetic studies of Duchenne muscular dystrophy families using the dystrophy families using the dystrophin cDNA].

The cDNA probes for the Duchenne muscular dystrophy (DMD) gene can detect deletions in over 50% of affected males and provide a highly accurate diagnostic test in the affected families. We present the results of our recent molecular genetic studies of five DMD families (six affected males and 16 non-affected family members) by using the dystrophin cDNA. Five molecular deletions were identified in the DNA samples of six affected males by studying abnormalities of Hind III and Bgl II fragments detected by the entire dystrophin cDNA. We could not find any abnormality in a family by the same DNA analysis. Deletion was found at two different loci; in two of six probands, it was located near 5' region of the gene seen by cDNA segment 1-2 a, while in the other three, near the center of the gene covered by cDNA segment 8. In three females of the five families, we detected novel fragments, the size of which was altered due to molecular deletion. These results greatly facilitated diagnosis of the carrier status in females. We studied also the dystrophin location in the biopsied skeletal muscle (femoral muscle) in a family, by means of indirect immunofluorescence staining using antiserum against dystrophin. Dystrophin was positive in the surface membrane of muscles from a non-affected brother, mother and sister, but it was absent in the muscle from a DMD proband.

[Clinical significance of dystrophin test for patients with various neuromuscular diseases--immunofluorescence and immunoblot analyses of dystrophin abnormalities].

The dystrophin test was performed on skeletal muscle specimens from 81 cases with various neuromuscular diseases by using two new monoclonal antibodies. The results were compared with those obtained by using four polyclonal antibodies. These monoclonal and polyclonal antibodies were raised against various portions of the dystrophin molecule. On immunohistochemical analysis, the two new monoclonal antibodies showed the same staining pattern as the four polyclonal antibodies. Non-specific immunostaining of the cytoplasm, often seen with polyclonal antibodies, was not observed with monoclonal antibodies. With the application of monoclonal antibodies, the connective tissue sometimes showed non-specific immunostaining which originated from the second fluorescent antibody. On immunoblot analysis, one of the two monoclonal antibodies, antibody 4-4 C 5, showed weak immunoreactivity, and the 400 kDa dystrophin band was not detected. Three cases out of 15 with Duchenne muscular dystrophy (DMD), and one case out of 3 with limb-girdle type muscular dystrophy which had previously been diagnosed on the basis of clinical data, were found to have non-dystrophin-related muscular dystrophy, and Becker muscular dystrophy (BMD), respectively. Three and two of five cases were diagnosed as DMD and BMD, respectively, though clinical diagnosis had not been possible because they were too young. Clinical diagnosis of congenital muscular dystrophy was confirmed in 9 patients by the dystrophin test. Only one of three certain DMD carriers had a so-called mosaic staining pattern. We conclude that all six antibodies are useful tools for the diagnosis of neuromuscular diseases, because of their high specificity for dystrophin.

[Molecular genetics of Duchenne/Becker muscular dystrophy].

The X-linked gene responsible for Duchenne muscular dystrophy (DMD)/Becker muscular dystrophy (BMD) encodes dystrophin, a high-molecular-weight cytoskeletal protein. The identification of the dystrophin gene through positional cloning, and the subsequent description of its protein product have opened several new fields of research and genetic diagnosis. Studies in our laboratory revealed that 26 out of 47 (55%) cases of DMD and nine out of 12 (75%) cases of BMD exhibited genomic deletion. The DMD phenotype is associated with mutations that shift the reading frame of the message, whereas the BMD phenotype is associated with mutations that maintain the reading frame. Immunofluorescence microscopy has established dystrophin's distribution on the plasma membrane of muscles. DMD patients demonstrate a lack of dystrophin on their muscle cell membrane, whereas BMD patients produce a limited amount of protein or abnormally sized protein. Extensive studies on dystrophin and the gene may lead to an understanding of the cause for this and may allow development of a rational treatment for DMD to be developed.

Intensive granulocyte and monocyte adsorption versus intravenous prednisolone in patients with severe ulcerative colitis: an unblinded randomised multi-centre controlled study.

BACKGROUND AND AIM: Several uncontrolled studies have reported on the efficacy of adsorptive depletion of peripheral blood granulocytes and monocytes/macrophages (GM) in patients with moderate or severe ulcerative colitis. This study was to compare the efficacy and safety of intensive GMA with intensive intravenous prednisolone in patients with severe ulcerative colitis. METHODS: Seventy patients with clinical activity index 10-23 were randomly assigned to intensive GMA with the Adacolumn, at 2 sessions/week in the first 3 weeks and then 1 session/week for up to 11 sessions (n = 35) or intravenous prednisolone, 40-60 mg/day for 5-10 days (n = 35). No patient received immunomodulators within 8 weeks prior to entry. Clinical response based on intention to treat was assessed at weeks 2, 6 and 12. RESULTS: Four patients in the prednisolone group and two patients in the GMA group discontinued in week 1. At weeks 2, 6 and 12, the remission (clinical activity index < or = 4) rates (%) in the GMA group were 17.1, 54.4, 74.3, respectively. The corresponding values in the prednisolone group were 25.7, 51.4 and 48.6. Further, at week 12, 27 patients (77%) in the GMA group and 5 patients (14%) in the prednisolone group were steroid free (P = 0.0076). In the GMA group, flushing and light-headedness were observed in 5 patients versus typical steroid side effects in 29 patients of the prednisolone group. CONCLUSIONS: In this clinical response to GMA was comparable or better than prednisolone. Further, the response to GMA was slower than to intravenous prednisolone, but was more sustainable than the latter.

Difference in the expression pattern of dystrophin on the surface membrane between the skeletal and cardiac muscles of mdx carrier mice.

We examined the expression of dystrophin by immunohistochemical and immunoblot analyses in the skeletal and cardiac muscles of Xmdx/X+ heterozygous mice, which were obtained by mating male mdx mice (Xmdx/Y) with female wild type mice (X+/X+). Dystrophin was expressed on the surface membrane in both muscles, but the mode of expression was different between the two muscles. In cardiac muscle, dystrophin positive and negative cells were present in roughly equal numbers intermingled in a mosaic pattern; this was considered to reflect the random inactivation of X-chromosomes in early development. In skeletal muscle, most of the surface membrane was dystrophin positive. There were little signs of fiber necrosis or regeneration, and serum creatine kinase levels were normal. We are at present of opinion that the predominance of dystrophin-positive area in skeletal muscle is due to intracellular diffusion of dystrophin.

Effects of sevoflurane and isoflurane on free radical formation in the post-ischaemic reperfused heart.

Sevoflurane has been reported to generate oxygen free radicals. We have investigated whether sevoflurane or isoflurane enhances oxygen free radical formation in the post-ischaemic reperfused heart. An isolated rat heart-lung preparation was used. Thirty male Wistar rats were allocated to four groups: control, no drug, 2.5% sevoflurane and 1.4% isoflurane. The heart was perfused initially at a cardiac output of 30 mL min-1 and a mean arterial pressure of 70 mmHg. Ten minutes after the start of perfusion, the heart was rendered globally ischaemic for 10 min by reducing the preload and afterload to zero. Then, the heart was reperfused for 10 min. At the end of reperfusion, the heart was freeze dried for 6 days. The perfusate blood was collected just before and just after ischaemia and at the end of reperfusion. The formation of hydroxyl radicals in perfusate blood and heart was measured with high-performance liquid chromatography using salicylic acid. Hydroxyl radicals react with salicylic acid, yielding 2,3-, 2,4-, 2,5- and 3,4-dihydroxybenzoic acid (DHBA). Before and after ischaemia, there were no significant differences in cardiac output, systolic pressure, heart rate and right arterial pressure among the groups. The concentrations of 2,3-, 2,4-, 2,5- and 3,4-DHBA in the perfusate blood after ischaemia and reperfusion were significantly higher than those before ischaemia in all groups. However, there were no differences in the DHBA levels among groups. This study indicates that sevoflurane and isoflurane do not enhance hydroxyl radical formation in the post-ischaemic reperfused heart.

Effects of melatonin and superoxide dismutase on free radical formation in the postischemic reperfused heart.

PURPOSE: Melatonin has been reported to protect against oxygen free radicals. We investigated whether melatonin or superoxide dismutase (SOD) would decrease hydroxyl radical concentration in the postischemic reperfused heart. METHODS: An isolated rat heart-lung preparation was used. Eighty-one male Wistar rats were allocated into control (no drug), S1 (SOD 400 U.ml(-1)), S2 (SOD 2000 U.ml(-1)), M1 (melatonin 0.1 microg.ml(-1)), M2 (melatonin 1.0 microg.ml(-1)), M3 (melatonin 10 microg.ml(-1)), SM (SOD 400 U.ml(-1) and melatonin 1.0 microg.ml(-1)) groups. The heart was perfused initially at the cardiac output of 30 ml.min(-1) and the mean arterial pressure of 70 mmHg. Drugs were administered into the reservoir 7 min after the start of perfusion. Ten minutes after the start of perfusion, the heart was rendered globally ischemic for 10 min by reducing the preload and afterload to zero and then reperfused for 10 min. At the end of reperfusion, the heart was freeze-dried for 6 days. The perfusate blood was collected just before and after ischemia and at the end of reperfusion. The formation of hydroxyl radicals in perfusate blood and heart was measured with high-performance liquid chromatography using salicylic acid. Hydroxyl radicals react with salicylic acid, yielding 2,3-, 2,4-, 2,5-, and 3,4-dihydroxybenzoic acid (DHBA). RESULTS: Before and after ischemia, there were no significant differences among the groups in cardiac output, systolic pressure, heart rate, and right atrial pressure. The concentrations of DHBAs in the perfusate blood and heart after ischemia and reperfusion in all groups were significantly higher than those before ischemia. DHBAs in the heart of all drug-administered groups were significantly lower than those in the control group. In the perfusate blood, DHBAs in the S2 group were significantly lower than those in the control group. CONCLUSIONS: SOD and melatonin decrease hydroxyl radical concentration in the postischemic reperfused heart.