Development of an efficient production method for beta-mannosidase by the creation of an overexpression system in Aspergillus aculeatus.

AIM: To develop an overexpression system in Aspergillus aculeatus in order to establish an efficient overproduction method of beta-mannosidase (MANB). METHODS AND RESULTS: An overexpression plasmid for the manB gene, encoding A. aculeatus MANB, was constructed and introduced into A. aculeatus cells. The gene was overexpressed under an improved promoter containing 12 copies of Region III cis-elements of Aspergillus oryzae in the transformant, and it secreted 2.56 mg MANB ml(-1) in liquid culture, which obtained a 9.4-fold higher productivity than that achieved in an overexpression system in A. oryzae. Most of the secreted protein in the cultured medium of the transformed A. aculeatus was the overproduced enzyme. CONCLUSIONS: Aspergillus aculeatus with the introduced overexpression plasmid produced 2.56 mg MANB ml(-1) in cultured medium. The improved promoter with A. oryzae Region III functioned in A. aculeatus; thus the strain is an expectant host for recombinant protein productions. SIGNIFICANCE AND IMPACT OF THE STUDY: The overexpression system with the improved promoter in A. aculeatus brought the highest productivity of MANB reported to date. The expression system would be a strong bioindustrial tool for protein production.

Acid sphingomyelinase deficiency: cardiac dysfunction and characteristic findings of the coronary arteries.

Two sisters with type B Niemann-Pick disease (genotype: S436R/S436R) showed cardiac dysfunctions, not secondary to pulmonary disease, at the beginning of the third decade. In the younger sister, myocardial dysfunction was refractory to treatment, resulting in death. At autopsy, the distal branches of the coronary arteries showed narrowing of the arterial lumina due to swelling of the medial and intimal smooth-muscle cells. This is the first report describing characteristic findings of coronary arteries in type B Niemann-Pick disease.

Effects of acute preload reduction on myocardial velocity during isovolumic contraction and myocardial acceleration in pediatric patients.

We evaluated the effects of acute preload reduction with inferior vena cava (IVC) occlusion on myocardial velocities during systole (Sa), early (Ea) and late (Aa) diastole, isovolumic contraction (IVV), and myocardial acceleration (IVA) measured by tissue Doppler imaging (TDI) in pediatric patients. A total of 22 patients (5 +/- 3 years) were studied: 9 patients (4 +/- 3 years) with Kawasaki disease, 8 patients (6 +/- 3 years) with atrial septal defect and right ventricular (RV) volume overload, and 5 patients (5 +/- 4 years) with pulmonary stenosis and RV pressure overload. Using TDI, Sa, Ea, Aa, IVV were recorded at the base of the RV free wall from a four-chamber view. IVA was calculated by dividing IVV by the time interval from onset of IVV to the time at peak velocity of this wave. In each group, IVC occlusion caused significant decreases in peak Sa, peak Ea, and peak Aa (p < 0.05). However, IVV and IVA did not change during acute preload reduction. This study demonstrated the effects of acute preload reduction on TDI velocities. In contrast to peak Sa, peak Ea, and peak Aa, IVV and IVA were unaffected by preload within a physiological range.

Posterior descending coronary artery flow reserve assessment by Doppler echocardiography in children with and without congenital heart defect: comparison with invasive technique.

To evaluate whether transthoracic Doppler echocardiography can reliably measure coronary flow velocity and coronary flow velocity reserve (CFVR) in the posterior descending coronary artery (PD) in children, we examined 32 patients who had congenital heart disease (ventricular septal defect in 10, tetralogy of Fallot in 6, tricuspid atresia in 3, double-outlet right ventricle in 2, patent ductus arteriosus in 2, and aortic valve stenosis in 2) and 7 patients who had a history of Kawasaki disease without stenosis or aneurysm formation of the coronary artery. Average peak flow velocity (APV) in the PD was measured by transthoracic Doppler echocardiography at the time of intracoronary Doppler study. CFVR was defined as the ratio of hyperemic to basal APV. Clear envelopes of basal and hyperemic APV in the PD were obtained in 23 of 32 patients by transthoracic Doppler echocardiography. APV obtained from transthoracic Doppler echocardiography correlated highly with that from the Doppler guidewire method (r=0.91). The mean difference between transthoracic Doppler echocardiography and the Doppler guidewire method was 0.1+/-2.9. There was an excellent correlation between transthoracic Doppler echocardiography and the Doppler guidewire method for the measurements of CFVR (r=0.84). The mean difference between transthoracic Doppler echocardiography and Doppler guidewire was -0.016+/-0.198. Noninvasive measurement of coronary flow velocity and CFVR in the PD using transthoracic Doppler echocardiography accurately reflects invasive measurement of coronary flow velocity and CFVR by the Doppler guidewire method in pediatric patients with various heart diseases.

Changes in pulmonary venous flow patterns in patients with ventricular septal defect.

We studied pulmonary venous (PV) flow patterns using Doppler echocardiography in 26 patients with ventricular septal defect less than 3 years of age. Fifteen patients had moderate or severe symptoms, and the remaining 11 had no significant symptoms. Peak velocity of PV diastolic flow and flow velocity integral of PV diastolic flow in the symptomatic patients were significantly larger than those in either asymptomatic patients or the normal controls. The ratio of PV diastolic flow velocity to PV systolic flow velocity and the ratio of flow velocity integral of PV diastolic flow to that of PV systolic flow in the symptomatic patients were significantly larger than those in either asymptomatic patients or the normal controls. The ratio of PV diastolic flow velocity to PV systolic flow velocity as well as the ratio of flow velocity integral of PV diastolic flow to that of PV systolic flow correlated with V wave in left atrial or pulmonary capillary wedge pressure and indexes of left ventricular mass and left atrial volume. We conclude that the abnormal pulmonary venous flow patterns in ventricular septal defect might be associated with large left-to-right shunting and left atrial pressure V wave.

An exonic mutation of the GH-1 gene causing familial isolated growth hormone deficiency type II.

A heterozygous base change was identified in exon 3 of the growth hormone (GH)-1 gene in a Japanese family with autosomal dominant GH deficiency. All of the patients from this family had a heterozygous G to T transversion at the first 5'-site nucleotide of exon 3. Analysis of the GH-1 cDNA, synthesized from lymphoblasts of the patients, revealed an abnormal shorter transcript as well as a normal-sized transcript. Direct sequencing of this abnormal transcript showed that the transcript completely lacked exon 3. In familial isolated GH deficiency (IGHD) type II, several heterozygous mutations have been reported at the donor splice site in intron 3 of the GH-1 gene or inside intron 3, which causes aberrant GH messenger RNA splicing, resulting in the deletion of exon 3. This deletion causes a lack of amino acid residues 32-71 in the mature GH protein. This mutant GH is well-known to exert a dominant negative effect on the secretion of mature normal GH protein. Thus, in the subject family, a heterozygous G-to-T transversion at the first nucleotide of the exon 3 deletes exon 3 in mature GH mRNA and causes GH deficiency. The present authors suggest that the first nucleotide of exon 3 is critical for the splicing of GH-1 mRNA.

[A case of carbamazepine intoxication with alpha coma and status epilepticus].

We report a 14-year-old girl with carbamazepine intoxication who developed alpha coma and status epilepticus. She fell into deep coma and developed frequent generalized convulsions. The EEG during coma showed diffuse alpha activity predominantly in the frontal area. Serum carbamazepine concentration was 42.8 micrograms/ml. The convulsions were suppressed by diazepam only transiently, and by midazolam completely. Although half a day had passed since carbamazepine ingestion, we could wash out much of drug remnants by gastric lavage. Thereafter, the serum concentration of carbamazepine decreased efficiently and the patient recovered dramatically without complication. Early diagnosis and appropriate treatments should improve the prognosis of carbamazepine intoxication.

Thyroid dysgenesis caused by PAX8 mutation: the hypermutability with CpG dinucleotides at codon 31.

We identified a novel mutation (CGC to T GC) at codon 31 of the Paired box 8 gene, an important transcription factor in the development of the thyroid gland. Mutations at this codon have been independently reported in 2 cases (CGC to CA C). These transitions are considered typical CpG-consequence mutations and account for hypermutability at this position.

Evidence for the association of ultraviolet-C and H(2)O(2)-induced apoptosis with acid sphingomyelinase activation.

Ceramide appears to be a potent second messenger implicated in the regulation of diverse cellular processes such as cell growth and differentiation, gene transcription, ligand binding, and cell death. Environmental stress-induced apoptosis is believed to be associated with the sphingomyelin degradation pathway, which generates ceramide as a second messenger in initiating the apoptosis response. To date, two distinct sphingomyelinases, a lysosomal acid sphingomyelinase (ASM), which is deficient in patients affected with types A and B Niemann-Pick disease (NPD), and a neutral, magnesium-dependent sphingomyelinase (NSM), are candidate enzymes which respond to apoptotic stimulations and cause sphingomyelin hydrolysis and subsequent ceramide generation. Using Epstein-Barr virus (EBV)-transformed lymphoblast cells from type A NPD patient which have defined splicing site mutation in the ASM gene, we showed that ASM-deficient cells were defective in ultraviolet-C (UV-C) and hydrogen peroxide (H(2)O(2)) induced apoptosis. As another induction of apoptosis, we exposed this cell line to serum starvation which influences to p53 expression and leads to apoptosis. There were no differences by the degree of apoptosis between ASM-deficient lymphoblast cells and normal lymphoblast cells. These results are evidence that ASM plays one of the important roles in apoptosis induction by UV-C and H(2)O(2).

Hypersomnia due to acute disseminated encephalomyelitis in a 5-year-old girl.

A 5-year-old girl suffering from hypersomnia due to acute disseminated encephalomyelitis (ADEM) is reported. Brain CT revealed a large low-density lesion involving the lentiform nucleus, posterior limb of the internal capsule, thalamus, posterior hypothalamus and midbrain in the left side. She was treated with intravenous dexamethazone. After the initial dose of dexamethazone, hypersomnia was dramatically and rapidly improved. A later brain CT study disclosed that the lesion in the brain disappeared. The brain lesion in this case involved the waking center in the brain, which was described by Von Economo. We concluded that hypersomnia in this case was due to ADEM involving the neural mechanism for maintaing wakefulness, probably in the thalamus and posterior hypothalamus. Repeat all night polysomnograpy in this case disclosed prolonged total sleep time and increased amount of stage 3-4 sleep in the hypersomniac state.

Body mass index can predict left ventricular diastolic filling in asymptomatic obese children.

To examine the effects of body mass index on left ventricular diastolic function, flow velocity patterns of the pulmonary vein and mitral valve were measured by pulse Doppler echocardiography in 21 asymptomatic obese children and were compared with those of an age-matched control population. The degree of obesity was calculated as (actual body mass index/ideal body mass index -1) x 100. The pulmonary venous flow indexes were peak systolic (S) and diastolic (D) velocities and peak D/S. The mitral inflow indexes were peak velocities of early diastole (E) and atrial contraction (A) and peak E/A. The pulmonary venous flow velocity pattern in obese patients was characterized by unchanged peak S, decreases in peak D (43 +/- 7 vs 51 +/- 8, p < 0.01) and peak D/S (0.98 +/- 0.19 vs 1.29 +/- 0.20, p < 0.01), suggesting the reduction in the early diastolic filling. The peak D/S decreased significantly with an increase in the percentage body mass index (r = -0.84, p < 0.01). In contrast to the pulmonary venous flow pattern (peak D > peak S) as seen in normal controls, all of the obese patients with > 70% over body mass index had abnormal pulmonary venous flow velocity patterns (peak D < peak S). The mitral flow velocity pattern in obese patients was also characterized by a decrease in early diastolic filling. However, these indices did not correlate with an increase in the percentage over body mass index. This study suggests that body mass index predicts the abnormality of left ventricular diastolic filling assessed by pulmonary venous flow patterns.

A new locus for a dominant form of multinodular goiter on 3q26.1-q26.3.

A mass screening program for congenital hypothyroidism has markedly improved prognosis of children with congenital hypothyroidism and also revealed several cases with unknown pathogenesis. We here report two independent Japanese multigeneration families with multinodular goiter (MNG) with euthyroidism and with high TSH. The propositi, 3- and 8-year-old girls in two families, were found during a mass screening. An autosomal dominant pattern of inheritance was suggested in both families. The clinical examinations suggested impaired hormonogenesis but discarded known defects in iodine transport, organification, deficiency of hydrogen peroxide, and thyroid peroxidase. Linkage analysis of the two families including 10 members each using 343 microsatellite markers mapped a single locus independently at D3S1618 (theta = 0) on 3q26.1-q26.3 with a two-point LOD score 3.62 (1.81 for each family) and multipoint LOD score of 3.61 (1.80 for each family). Haplotype inspection delimited an 18-cM interval between D3S1565 and D3S3686.

Ala/Thr60 variant of the Leydig insulin-like hormone is not associated with cryptorchidism in the Japanese population.

BACKGROUND: Leydig insulin-like hormone (Insl3), a member of the insulin-like superfamily, is specifically expressed in Leydig cells of fetal and postnatal murine testis. Recently, the absence of the Insl3 gene has been reported to result in bilateral cryptorchidism in male mice and it has been suggested that mutations of the INSL3 gene may cause cryptorchidism in humans. METHODS: We sequenced the INSL3 gene from five Japanese patients with sporadic bilateral cryptorchidism. Patients' genome DNA was prepared from blood leukocytes. Two exons of the INSL3 gene were amplified by polymerase chain reaction and were sequenced directly. A restriction fragment length polymorphism assay was performed on 70 control samples for analysis of polymorphism. RESULTS: Three of five cases had a heterozygous single-base change, a G to A transition at position 178 of the INSL3 gene, which predicts an alanine (GCC) to threonine (ACC) change at codon 60 (designated A60T). However, the A60T mutation was also found in the normal Japanese population at an allele frequency of 26%, which suggests that this mutation is a common polymorphism and is not associated with the occurrence of cryptorchidism. CONCLUSIONS: No mutation has been found in the INSL3 gene from Japanese patients with idiopathic cryptorchidism. We did find the A60T polymorphism, which was not associated with the occurrence of cryptorchidism.

Cardiorespiratory exercise capacity and its relation to a new Doppler index in children previously treated with anthracycline.

The purpose of this study was to assess the exercise capacity of patients treated with anthracycline and to evaluate the relation between the exercise capacity and a new Doppler index. The study patients consisted of 70 subjects: 41 healthy subjects and 29 who had been treated with various cumulative doses of anthracycline (range 45 to 873 mg per body surface area). The following conventional echocardiographic parameters were measured: rate-corrected mean velocity of fiber shortening (mVcfc), end-systolic wall stress (ESS), stress-velocity index, and early and late diastolic mitral inflow velocities and their ratio. A new Doppler index, the Tei index, was calculated as the sum of isovolumic contraction time and isovolumic relaxation time divided by the ejection time. Peak oxygen uptake (pVo(2)) and anaerobic threshold (AT) were measured during an upright bicycle exercise test. The pVo(2) and AT in the patient group were significantly lower than those in the control group (pVo(2): 22.0 +/- 3.7 versus 28.5 +/- 7.1 mL/min/kg; AT: 12.7 +/- 1.9 versus 17.3 +/- 4.3 mL/min/kg, respectively; P <.01). There were no significant differences in the mVcfc, ESS, stress-velocity index, E wave, A wave, or E/A wave ratio between the two groups. However, the mean Tei index of the patients was significantly greater than that of the controls (0.41 +/- 0.11 versus 0.33 +/- 0.04, P <.01). The pVo(2) and AT decreased significantly with an increase in the Tei index (r = -0.64 and -0.60, respectively; P <.01). A weak positive correlation was found between the AT and E/A wave ratio (r = 0.54, P <.05). However, no significant correlations were seen between the exercise parameters and the mVcfc, ESS, stress velocity index, or transmitral velocities. Our findings suggest that cardiopulmonary exercise testing revealed an inverse correlation between exercise capacity and the Tei index.

In situ morphometric analysis of left and right ventricles in fetal rats: changes in ventricular volume, mass, wall thickness, and valvular size.

We studied morphological changes in the left and right ventricles of fetal rats in late-gestation using rapid whole-body freezing technique. Pregnant Wistar rats (term, 21.5 day) were immediately frozen in liquid nitrogen on 17-, 18-, 19-, 20-, and 21-day of gestation. The frozen fetal hearts were serially sectioned with a sliding microtome and photographed. The ventricular volume, mass, wall thickness, and area of valvular orifice were measured on the photographs. During the study period, the left and right ventricular volumes increased very rapidly (9.9-fold and 7.6-fold, respectively) compared with the increase in the body weight (4.0-fold); the volumes divided by body weight increased linearly. Left and right ventricular masses also rapidly increased (5.9-fold and 5.0-fold, respectively). Mass/volume ratios for the two ventricles rapidly decreased. The wall thicknesses divided by body weights rapidly decreased with the progression of the gestational age. The left and right ventricles at 17 day of gestation have relative hypertrophy and relatively large valvular orifices as compared with those in terminal gestation. The improvement of the relative hypertrophy of the ventricles may indicate the morphological and functional maturation of the fetal heart.

Status of platelet collection and platelet transfusion.

Platelet product derived from single donor plateletpheresis is required to reduce the risks of adverse reactions by blood transfusion. The objectives of this study are to evaluate the status of platelet collection and its efficacy by various kinds of plateletpheresis equipment and to assess the achievement of platelet transfusion by platelet product derived from a single donor. Since the blood centers have introduced some kinds of efficient plateletpheresis equipment, large units of platelet products have been supplied mainly for the patients. Amicus and CCS might be preferable plateletpheresis machines because of their collection efficiencies and wider indication for donors. The average number of donors of platelet product per patient has recently reached nearly 1.0, and around 90% of patients have received platelet product derived from a single donor in the recent several years. However, platelet transfusion derived from a single donor has not yet been completely achieved. Each regional blood center should seriously consider the efficacy of each plateletpheresis equipment and arrange the equipment to collect platelets more effectively to achieve platelet transfusion from a single donor.