Mutation analysis of left-right axis determining genes in NOD and ICR, strains susceptible to maternal diabetes.

BACKGROUND: Genetic background of the fetus contributes to the pathogenesis of congenital malformation after teratogen exposure. Such contribution is illustrated in left-right axis malformations observed in the F1 offspring of nonobese diabetic (NOD) mouse dams and sires from different strains. When sires of the NOD, ICR, or C57BL/6J were mated with NOD dams, incidence varied depending on the fetal genotype, with 65% in NOD x NOD, 24% in NOD x ICR, and 7% in NOD x C57BL/6J. METHODS: As a first step in elucidating the molecular basis of the interstrain differences in susceptibility to situs defects, we compared genomic sequences of six genes HNF3beta, Acvr2b, Nodal, ZIC3, Lefty1, and Smad2, which are involved in the normal development of left-right axis among NOD, ICR, and C57BL/6J strains. RESULTS: The outbred strain ICR had 1) a 0.2-kb insertion in the putative promoter region of the isoform E of HNF3beta together with a G to A change that could create a potential splice acceptor in the exon 3 of HNF3beta (gene frequency P = 0.36), 2) five single base substitutions within the 5' controlling element and a proline to serine substitution (P2S) of Lefty1 (P = 0.77), and 3) a tyrosine to histidine substitution within the prodomain of Nodal (P = 0.48). The inbred strain NOD had the same G to A change as ICR and a three-base deletion in the putative promoter of isoform E of HNF3beta. CONCLUSIONS: We suggest that sequence variations in HNF3beta, Lefty1, and Nodal might account, in part, for the interstrain differences in susceptibility to situs abnormalities among the offspring of diabetic dams.

Human homolog of the mouse imprinted gene Impact resides at the pericentric region of chromosome 18 within the critical region for bipolar affective disorder.

Several mapping studies of families with multiple individuals who have bipolar affective disorder (BPAD) have demonstrated possible linkage of the trait to the pericentric region of chromosome 18 (18cen). Currently, the large size of the critical interval defined by these studies makes effective selection of candidate genes formidable. However, documentation of 18cen-linked families in which a parent-of-origin effect was observed in the transmission of the BPAD trait provides a clue to the nature of the putative gene; it may be imprinted. In the present study, we cloned IMPACT, the human homolog of the mouse imprinted gene Impact and mapped it to 18cen within the critical interval for BPAD. Human IMPACT encodes a protein with 320 amino acids and is expressed at high levels in the brain. Since only a small number of imprinted genes are estimated to be present in the entire genome, very few imprinted genes would be expected to be present in this particular chromosomal region. Hence, IMPACT represents a candidate gene for BPAD susceptibility. Alternatively, other as yet unknown imprinted gene(s) adjacent to IMPACT could contribute to the BPAD trait, since multiple imprinted genes may occasionally form clusters. Localization of human IMPACT at 18cen in this study defines a promising target region in which to search for putative BPAD genes.

Fluorescence-based DHPLC for allelic quantification by single-nucleotide primer extension.

We have investigated the possibility of determining quantitatively the alleles of binary DNA polymorphisms by single-nucleotide primer extension (SNuPE) and fluorescence-based DHPLC. Using a polymorphism of interest to our group, ROX-labeled dideoxy CTP (ROX-ddCTP) was incorporated at the 3' end of the primer annealed to the template adjacent to the polymorphic site. The primer extension product was then resolved from the unincorporated dye terminator by ion-pair reversed-phase liquid chromatography. The signal intensity of incorporated ROX-ddCTP correlated well over one order of magnitude with the relative amount of the C-allele present in the genomic DNA template. We conclude that SNuPE, when combined with fluorescence-based DHPLC, can accurately determine the relative molar proportion of one allele in total DNA.

Imprinting of human GRB10 and its mutations in two patients with Russell-Silver syndrome.

Documentation of maternal uniparental disomy of chromosome 7 in 10% of patients with Russell-Silver syndrome (RSS), characterized by prenatal and postnatal growth retardation and dysmorphic features, has suggested the presence of an imprinted gene on chromosome 7 whose mutation is responsible for the RSS phenotype. Human GRB10 on chromosome 7, a homologue of the mouse imprinted gene Grb10, is a candidate, because GRB10 has a suppressive effect on growth, through its interaction with either the IGF-I receptor or the GH receptor, and two patients with RSS were shown to have a maternally derived duplication of 7p11-p13, encompassing GRB10. In the present study, we first demonstrated that the GRB10 gene is also monoallelically expressed in human fetal brain tissues and is transcribed from the maternally derived allele in somatic-cell hybrids. Hence, human GRB10 is imprinted. A mutation analysis of GRB10 in 58 unrelated patients with RSS identified, within the N-terminal domain of the protein, a P95S substitution in two patients with RSS. In these two cases, the mutant allele was inherited from the mother. The fact that monoallelic GRB10 expression was observed from the maternal allele in this study suggests but does not prove that these maternally transmitted mutant alleles contribute to the RSS phenotype.

Regional cerebral blood flow in lacunar infarction.

Ten cases of lacunar infarction, 10 cases of nonlacunar cerebral thrombosis, and 8 healthy controls who did not have risk factors of cerebrovascular diseases were studied. Subcortical cystic infarctions with a diameter of less than 1.5 cm were classified as lacunar infarction and the other cerebral thrombosis were classified as nonlacunar cerebral thrombosis. Cerebral blood flow examination by Xenon computed tomography (CT) method was performed within 14 days after the onset of stroke. Stable Xenon was inhaled for 3 minutes and CT scan was taken once before the inhalation, 3 times during the inhalation, and 5 times in the washout phase. Regional blood flows in the infarcted area, around the infarcted area, and in the cerebral cortex and the cerebral white matter where the influence of the infarction was considered to be little were measured before and after intravenous injection of 17 mg/kg acetazolamide. In the lacunar infarction, the blood flow in the cerebral cortex where the influence of the infarction was considered to be little was decreased and the cerebrovascular dilatory reserve capacity in the cerebral cortex and the cerebral white matter was decreased. Arteriolosclerosis is considered to be the basic cause of lacunar infarction.

Dysphasia accompanied by periodic lateralized epileptiform discharges.

We encountered a 67-year-old man who presented with repetitious dysphasia accompanied by periodic lateralized epileptiform discharges (PLEDs) on the electroencephalogram. A good correlation was established between the dysphasia and the PLEDs. A persistent partial seizure accompanied by PLEDs originating in the left hemisphere presented with dysphasia clinically. None of the previously reported patients with epileptic dysphasia had accompanying PLEDs. The administration of carbamazepine was successful to terminate the seizure, however valproate was not. This case indicates that PLEDs may produce significant, however reversible, functional damage, and may advance our understanding of the pathophysiology of epileptic dysphasia.

Effects of sodium ozagrel on hemostatic markers and cerebral blood flow in lacunar infarction.

The present study was designed to examine the effects of sodium ozagrel on hemostatic markers and cerebral blood flow in lacunar infarction. Ten cases of lacunar infarction in which sodium ozagrel was given (administered group), 10 cases of lacunar infarction in which sodium ozagrel was not given (nonadministered group), and 10 age-matched controls in which cerebrovascular diseases were absent but risk factors were similar to those of the patients (control group) were studied. Intravenous infusion of 80 mg of sodium ozagrel was done twice a day for 2 weeks. Platelet factor 4, beta-thromboglobulin, and fibrinopeptide A were significantly higher in the administered and nonadministered groups than in the control group at the time of admission. Platelet factor 4, beta-thromboglobulin, fibrinopeptide A, and thromboxane B2 were decreased significantly by the administration of sodium ozagrel. The blood flow in the cerebral cortex was significantly lower in the administered and nonadministered groups than in the control group. The blood flows around the infarcted area, in the cerebral cortex, and in the cerebral white matter were significantly increased by the administration of sodium ozagrel. Sodium ozagrel is considered to decrease platelet aggregation and increase cerebral blood flow by decreasing thromboxane A2, which has a platelet-aggregating and a vasoconstricting action. Sodium ozagrel is considered to be effective in the acute phase of lacunar infarction.

Laboratory examinations correlated with severity of dementia.

Acetylcholine esterase, alpha 1-antichymotrypsin, homovanillic acid (HVA), 3-methoxy-4-hydroxy-phenylenglycol (MHPG), norepinephrine, dopamine, 5-hydroxy-indolacetic acid (5-HIAA), and gamma-aminobutyric acid (GABA) in the serum and cerebrospinal fluid (CSF) were quantified. Positive wave with the latency about 300 msec (P300) and electroencephalography (EEG) were examined in 10 patients with Alzheimer's disease, 10 patients with vascular dementia, and 10 age-matched healthy controls. Serum alpha 1-antichymotrypsin concentrations were significantly higher in the Alzheimer's disease group than in the vascular dementia and healthy control groups. Homovanillic acid concentrations in CSF were significantly lower in the vascular dementia group than in the Alzheimer's disease and the healthy control groups. A significant positive correlation was present between the mini-mental state examination (MMSE) score (normal range of 24 to 30) and the acetylcholine esterase concentration in the CSF. Significant negative correlations were present between the MMSE score and the P300 latency, between the MMSE score and the MHPG concentration in the CSF, between the MMSE and the norepinephrine concentration in the CSF, and between the MMSE score and the dopamine concentration in the CSF.

Crow-Fukase syndrome: a case associated with vasospastic angina.

The patient is a 60-year-old man who developed numbness of the extremities, paralysis, hyperpigmentation of the skin, hypertrichosis, anasarca and chest pain at the age of 58 years. The diagnosis of Crow-Fukase syndrome was made and myeloma was not found. Prednisolone therapy was effective but chest pain reappeared every morning when prednisolone was tapered to 30 mg alternate day. Coronary arteriogram showed no stenosis but administration of acetylcholine into the coronary artery produced ST elevation in electrocardiogram, chest pain and coronary artery stenosis which were relieved by administration of nitrates into the coronary artery.

Spread of herpes simplex virus type-1 (Miyama +GC strain) to the central nervous system after intraperitoneal inoculation: the role of the myenteric plexus of the gut.

The pathways taken by the HSV-1 virus after intraperitoneal (i.p.) inoculation were studied in 5-week old male C3H/HeN mice injected with 1 x 10(4) PFU (100 LD50) or 5 x 10(5) PFU (5000 LD50) of HSV-1 (Miyama +GC strain). At the higher dosage (5 x 10(5) PFU), HSV-1 began replicating in the adrenal from the first day, then in the gut and thoracic portion of the spinal cord by the third day, and in the brainstem by the fourth day, as shown by the titers of the virus in these organs. By immunoperoxidase staining HSV-1 was localized in a necrotic area of the adrenal, the myenteric plexus of the gut, the intermediolateral columns of the thoracic cord, and the vagus nerve nuclei of the medulla oblongata. In the low dose mice (1 x 10(4) PFU), HSV-1 was not isolated from the adrenal or thoracic segment of the spinal cord from the time of inoculation until the time of death. It was, however, isolated from the gut on days 4-6 and from the brainstem by day 5. HSV-1 was never isolated from the blood of either group at any time. The localizations of viral replication suggest that in the mice inoculated with 1 x 10(4) PFU, HSV-1 spreads to the brainstem via the vagal nerves after replication in the myenteric plexus of the gut. In the mice given the higher dose, localizations suggest not only the above route, but also that the virus spread to the intermediolateral columns of the spinal cord after replicating in the adrenal.