Non-radioactive screening of DNA polymerases by using oligo r(A-U) as primer/template for reverse transcriptase activity.
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DNA Primers↗
Biomedical subjects
K Hanaki
Publications and source records attributed to K Hanaki.
Hybridization-AT-tailing (HybrAT) method for sensitive and strand-specific detection of DNA and RNA.
delta Tth DNA polymerase catalyzed polymerization of dATP and dTTP into a high-molecular-weight d(A-T) copolymer using oligo-d(A-T) as the template/primer (Hanaki et al., Biochem. Biophys. Res. Commun. 244, 210-219). Taking advantage of this reaction, we developed a highly sensitive method for strand-specific detection of DNA or RNA. The probe consisted of a 40- to 50-base-long complementary sequence on the 5' side and 10 repeats of AT on the 3' side. After hybridization using the 5' side, the 3' side AT repeat region was elongated by delta Tth DNA polymerase in the presence of the dATP, dTTP, and digoxigenin (dig)-11-dUTP. The elongation condition was 52-62 degrees C for 3 h. The method named HybrAT (hybridization-AT-tailing) was at least 100-fold more sensitive than the conventional hybridization with 5' end dig-11-dUTP labeled probe.
Adenine↗
Two different reactions involved in the primer/template-independent polymerization of dATP and dTTP by Taq DNA polymerase.
Taq and Tth DNA polymerases catalyzed polymerization of dATP and dTTP into poly d(A-T) without requiring added primer/template (Hanaki et al., Biochem. Biophys. Res. Commun. 238, 113-118), while the Stoffel fragment of Taq DNA polymerase and delta Tth DNA polymerase with respective deletions of ca. 290 and 250 N-terminal amino acids did not. The primer/template-independent polymerization appeared to proceed via two reactions, the slow process of formation of 16-19 nt long oligo d(A-T) without primer/template and the rapid process of elongation of the oligo d(A-T) by self-priming. As the former step was more sensitive to N-ethylmaleimide than the elongation reaction, probably the formation of the oligonucleotide preceded the elongation. But when the substrates were depleted, Taq DNA polymerase degraded the high molecular weigh d(A-T) polymer to the oligomers which were resistant to the further digestion by the 5'-->3' exonuclease activity of Taq DNA polymerase. Probably, the elongation and the degradation reactions proceeded simultaneously, the former process being faster than the latter in the presence of enough dATP and dTTP.
DNA↗
Inhibitory effects of modified oligonucleotides complementary to the leader RNA on the multiplication of mouse hepatitis virus.
Phosphorothioate oligonucleotides (PS-oligo) and PS-oligos with cholesterol conjugates (ChPS-oligo) complementary to the leader RNA of strain JHM of mouse hepatitis virus (JHMV) were more effective inhibitors of viral multiplication than natural oligodeoxynucleotides (PO-oligo) in JHMV-infected DBT cells. PS- and ChPS-oligos were 1,000 times more potent than unmodified PO-oligo. No significant difference was observed in the inhibitory efficiency between PS-oligo and ChPS-oligo. Sequence-dependent inhibition of viral multiplication was shown at low concentrations (0.001-0.1 M) of antisense PS-oligo and ChPS-oligo. Phosphorothioate oligodeoxycytidine, PS-(dC)20, and PS-(dC)20 with cholesterol conjugates, and PS- and ChPS-oligo which have no significant homology to the JHMV sequences, showed inhibitory effects on JHMV multiplication at concentrations higher than 0.5 M. These results showed that PS-oligo and ChPS-oligo were more potent than PO-oligo in the inhibition of JHMV multiplication, and that PS-oligo and ChPS-oligo may inhibit JHMV multiplication by two different mechanisms, that is by sequence-dependent and sequence-independent manners.
5' Untranslated Regions↗
cis Elements required for high-level expression of unspliced Gag-containing message in Moloney murine leukemia virus.
The 441-nucleotide (nt) region (nt 5325 to 5766) around the splice acceptor (SA) site (nt 5491) was found to be necessary for high-level expression of gag-containing unspliced RNA of Moloney murine leukemia virus (M. Oshima, T. Odawara, T. Matano, H. Sakahira, K. Kuchino, A. Iwamoto, and H. Yoshikura, J. Virol. 70:2286-2295, 1996). Detailed genetic dissection of the 441-nt region revealed that the 5'-end 64 nt (nt 5325 to 5389) were necessary for high-level expression of the unspliced RNA when the spliced RNA was not produced, while the 3'-side 301 nt (nt 5466 to 5766) containing the SA site were necessary for producing spliced RNA. When the spliced RNA was produced, the unspliced RNA could be expressed at a high level even when the 5'-end 64 nt were absent. Probably the virus sequence ensuring the splicing could produce an RNA structure able to compensate for the function of the 5'-end 64-nt region responsible for the expression of the unspliced RNA.
3T3 Cells↗
Primer/template-independent synthesis of poly d(A-T) by Taq polymerase.
Taq DNA polymerase polymerized dATP and dTTP to poly d(A-T) without requiring added primer/template in the temperature range of 60-70 degrees C. Tth DNA polymerase also catalyzed the reaction, while delta Tth, Vent, Vent(exo-), Pfu, Ultma, BcaBEST, and KOD DNA polymerases did not. The reaction was distinct from the template-nonrequiring terminal deoxynucleotidyl transferase reaction which absolutely required primers.
DNA Nucleotidylexotransferase↗
Analysis of the steroidogenic acute regulatory protein (StAR) gene in Japanese patients with congenital lipoid adrenal hyperplasia.
Genomic DNA from 19 Japanese patients with congenital lipoid adrenal hyperplasia (lipoid CAH) representing 16 different families was examined to identify the genetic alterations of steroidogenic acute regulatory protein (StAR). Ten of 19 patients had a 46,XX karyotype and nine had a 46,XY karyotype. Six of the 46,XX patients have experienced spontaneous pubertal changes including breast development and irregular menstruation whereas none of the 46,XY subjects displayed pubertal changes. Eight different mutations were identified. Sixteen patients were either homozygotes or compound heterozygotes for the Q258X mutation. The seven other mutations identified were 189delG, 246insG, 564del13bp, 838delA, Q212X, A218V and M225T. The 189delG, 246insG, 546del13bp and Q212X mutants encode truncated proteins. COS-1 cells transfected with expression vectors encoding cDNAs for the mutant StAR proteins which affect the C-terminus, 838delA, A218V and Q258X, exhibited no steroidogenesis enhancing activity. However, the M225T mutant retained some steroidogenic activity. The patient with the M225T mutation had late onset of this disorder and some capacity to secrete testosterone in response to hCG. These findings suggest: (i) that the Q258X mutation can be used as a genetic marker for the screening of Japanese for lipoid CAH, (ii) that the C-terminus of StAR plays an important role in the protein's activity and (iii) that there are differences in the extent of functional impairment of the testis and ovaries in lipoid CAH.
Adrenal Hyperplasia, Congenital↗
Partial monosomy 10q and partial trisomy 9q with anal atresia due to maternal translocation: t(9;10)(q32;q26).
We report a case of a boy with a partial deletion of the long arm of chromosome 10 and partial duplication of the long arm of chromosome 9 due to maternal balanced translocation. The karyotype was 46,XY,-10, +der(10)t(9;10)(q32;q26)mat. Our patient had imperforate anus and as this finding is usually not observed in association with trisomy 9q, it suggests that partial monosomy of the long arm of chromosome 10 might be associated with anogenital anomalies.
Abnormalities, Multiple↗
Maternal perception of children's weight in relation to eating disorders.
Abnormal perception of body weight in girls has been reported previously (1), and distorted perception of body shape is cited as one of the pathogenetic factors in eating disorders such as anorexia and bulimia nervosa (2,3). Parent-child relationships should also be considered as one of the factors possibly contributing to the onset of the eating disorders (3,4). In the present paper we shall examine whether mothers perceive the weight of their sons and daughters correctly or not, and we shall also compare the results with the perceptions of the children themselves, which we have reported previously in this journal (1). Five outline drawings (-20% to 20% overweight), used in the previous study (1), were shown to the mothers, who were asked to select the drawings which they considered to represent the body weight of their sons (n = 130) and daughters (n = 125), who were aged 6 to 18 years. The mean body weights of the boys and girls in the study sample were 6.0% and 6.6% overweight, respectively. The mothers perceived the weight of their sons to be significantly (P < 0.01) less than the actual weight of these boys. The mean difference between the perceived weight and the actual weight was 10.4%. The mothers' perception of their daughters' weight was also 7.9% less than the actual weight (P < 0.05) (Fig. 1).
Adolescent↗
Inhibition of mouse hepatitis virus multiplication by phosphorothioate analogues of oligodeoxynucleotides complementary to the leader RNA.
Phosphorothioate oligodeoxynucleotides (PS-oligo) complementary to a leader RNA of mouse hepatitis virus (MHV) were more effective inhibitors of MHV multiplication than natural oligodeoxynucleotides (PO-oligo). Sequence-dependent inhibition of viral multiplication was shown at low concentrations (0.001-0.1 microM) of antisense PS-oligo. Phosphorothioate oligodeoxycytidine, PS-(dC)20 and PS-oligo, which has no significant homology to the MHV sequence, showed inhibitory effects on MHV multiplication at concentrations higher than 0.5 microM. These results showed that PS-oligo was more potent than PO-oligo in inhibition of MHV multiplication and that PS-oligo may inhibit MHV multiplication by two different mechanisms, that is, in sequence-dependent and -independent manners.
Animals↗
An infant with pseudohypoaldosteronism accompanied by cholelithiasis.
A case of an infant with very rare concurrence of pseudohypoaldosteronism and cholelithiasis is described. Aldosterone concentration was extremely high (64.6 nmol/l; normal 0.03-4.4 nmol/l) and fludrocortisone did not have favorable effects on hyperkalemia (7.4 mEq/l) and hyponatremia (124 mEq/l). A gallstone was found using ultrasonography at age 6 months, and it was extirpated surgically. The combination has not been reported previously. The study supports the previous hypothesis that cholelithiasis is found more often in infants with dehydration or electrolyte derangement.
Aldosterone↗
Excessive food aversion, compulsive exercise and decreased height gain due to fear of obesity in a prepubertal girl.
A case of a 7-year-old girl with a remarkable food aversion and excessive weight reduction caused by fear of obesity, which has been demonstrated in pubertal girls with symptoms partly similar to anorexia nervosa, is reported. Although the patient's weight was reduced to the upper limits of the normal range with diet and exercise, she reduced her food intake more strictly and did not at all eat food consisting of carbohydrates. Exercise was performed longer than before. Her weight continued to decrease and height velocity lowered from 6.0 to 4.1 cm/year (mean +/- SD of the age-matched normal girls: 5.5 +/- 0.74 cm/year). Her eating behavior was normalized without specific psychotherapy for anorexia nervosa. It is suggested that food aversion with weight loss and decrease in height gain due to fear of obesity may occur in prepubertal children as well as in adolescent girls.
Anorexia Nervosa↗
Assessment of subcutaneous fat using ultrasonography in the Ullrich-Turner syndrome.
Subcutaneous fat was measured using an ultrasonic method (SF-US) in girls with Ullrich-Turner syndrome (UTS) and in height and weight-matched controls. The method is not influenced by changes in cutaneous compressibility, which is frequently abnormal in UTS especially at a younger age. SF-US in the arms was significantly less in UTS patients (ulnar SF-US: P < 0.05; triceps SF-US: P < 0.005) than in the control girls. Results in suprailiac SF-US measurement was also similar (P < 0.05). Though the mean SF-US in the subscapular region was less in UTS patients than the control girls, there was no statistically significant difference. It is demonstrated that subcutaneous fat in UTS patients is less in their arms than in girls without UTS and that trunkal fatness is not increased but rather decreased in the UTS. The ratio of lean body mass to weight seems higher in UTS patients than in girls with similar weight and height.
Adipose Tissue↗
Brachydactyly type A-4 (Temtamy type) with short stature in a Japanese girl and her mother.
We report on a Japanese girl and her mother with brachydactyly. Their 2nd and 5th middle phalanges were short and the latter was fused with the distal phalanx in one of the patients. Length and shape of proximal and distal phalanges as well as metacarpals seemed normal. These findings are consistent with brachydactyly type 4-A, which is a rare subtype and has not been adequately documented. Short stature was reported only in some cases of brachydactyly type A-1, A-6, C, and E, but not in the other subtypes. Our patients with type A-4 brachydactyly also had short stature.
Abnormalities, Multiple↗
Salt-losing form of 21-hydroxylase deficiency accompanied by hypopituitarism in a boy.
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Adrenal Hyperplasia, Congenital↗
Carbohydrate-deficient glycoprotein syndrome in a girl with hypogonadism due to inactive follicle stimulating hormone.
We report a very rare case of a girl with hypogonadism probably due to inactive FSH. She had been diagnosed as carbohydrate-deficient glycoprotein syndrome. The 14-year-old patient had no secondary sexual characteristics with the 46,XX karyotype and estradiol (E2) levels were undetectable. Follicle stimulating hormone (FSH) levels were extremely high and responded exaggeratedly to luteinizing hormone-releasing hormone (LH-RH). The E2 levels, on the other hand, were elevated after stimulation with human menopausal gonadotropin (75 units/day) for 5 days, suggesting biologically inactive FSH in the circulation. Abnormal structure in the glycoprotein may be possibly related with hypogonadism.
Adolescent↗
Long QT syndrome with insulin-dependent diabetes mellitus: contiguous gene syndrome on chromosome 11p.
Long QT syndrome (Romano-Ward syndrome) and insulin-dependent diabetes mellitus (IDDM) have been documented as being linked with gene(s) on chromosome 11p although concurrence of the two disorders has not been reported. Our case is a 13-year-old boy with Romano-Ward syndrome accompanied by IDDM. The long QT syndrome seemed to be transmitted in an autosomal-dominant mode because the Q-T intervals of his father and paternal grandfather were longer than normal. There was no family member with an abnormally high level of blood glucose except the patient. The human leucocyte antigen (HLA) haplotypes of the patient and the father were DR4/DR9 and DR2/DR9, respectively. This study suggests that in our patient IDDM, as well as Romano-Ward syndrome, is linked with chromosome 11p in the presence of HLA-DR4. The results support the previous study that chromosome 11p encodes a gene implicated in HLA-DR4-dependent diabetes susceptibility.
Child↗
Responses of plasma vasoactive intestinal polypeptide to methacholine and exercise loading in children and adolescents with bronchial asthma.
Responses of plasma vasoactive intestinal polypeptide (VIP) to methacholine inhalation and to exercise loading were studied in asthmatic patients to clarify a significant role of the peptide. The mean of basal VIP in asthmatics was not significantly different from the normals. The levels were increased after FEV (1.0) (forced expiratory volume in a second) decreased to 80% of the baseline following methacholine inhalation and were returned to values similar to the baseline when FEV (1.0) recovered to 100%. Thirty minutes after the exercise, mean VIP was significantly lower in patients with the lowest FEV (1.0). These results suggest that VIPergic system contributes, at least partly, to relax constricted bronchioles in some patients with bronchial asthma.
Adolescent↗