Identification of N-homocysteinylated apolipoprotein AI in normal human serum.

BACKGROUND: In human serum, a portion of homocysteine (Hcy) exists as an N-linked form to the epsilon-amino group of protein lysine residues. N-homocysteinylated proteins differ structurally and functionally from native proteins. The present study strives to develop detection and potential semi-quantification methods for N-homocysteinylated apolipoprotein AI (N-Hcy-apoAI) in human serum. METHODS: Serum treated with or without cysteamine was supplied to isoelectric focusing (IEF) followed by an immunoblot using an anti-apoAI antibody. Cysteamine treatment increased the isoelectric point for N-Hcy-apoAI, but not for unmodified apoAI, due to the presence of -SH group(s) derived from Hcy and the absence of a cysteine residue in the apoAI molecule. N-Hcy-apoAI was semi-quantified from the scanned immunoblot pattern via a computer. RESULTS: After cysteamine treatment, N-Hcy-apoAI in the serum was identified by IEF at the position with a higher pI value compared with intact apoAI. The reproducibility (between assays) of the semi-quantification method was 19.1% CV (coefficient of variation) for an average ratio 5.9% of N-Hcy-apoAI to the whole apoAI in the serum. Approximately 1.0-7.4% of apoAI was N-homocysteinylated in the serum obtained from 27 healthy subjects. Neither the ratio of N-Hcy-apoAI nor its concentration, calculated by total apoAI concentration, indicated correlation with the so-called total (free and S-linked) Hcy concentration. CONCLUSIONS: We directly found that a portion of apoAI in the serum undergoes homocysteinylation in an N-linkage manner, and used this to develop a potential semi-quantification method for N-Hcy-apoAI.

Plasma nitric oxide products correlate with cardiac index of congenital heart disease.

We wished to determine the relationship between circulating levels of nitric oxide (NO) and cardiac index (CI) in children with congenital heart diseases. We measured the plasma levels of nitrate/nitrite (NO(x)), the stable end products of NO production as well as tumor necrosis factor-alpha (TNF-alpha), atrial natriuretic peptide (ANP), and brain natriuretic peptide in relation to various parameters determined simultaneously. The plasma NO(x) levels correlated negatively with CI (r = -0.541, p < 0.05). No correlation was observed between NO(x) and cardiac output. TNF-alpha correlated with NO(x) levels (r = 0.593, p < 0.005) but not with either CI or cardiac output. Plasma levels of ANP and TNF-alpha were higher in atrial septal defect than those in the control group (p < 0.001 and p < 0.05, respectively). Elevated plasma NO(x) could explain the increased basal release of endothelial NO due to high pulmonary blood flow. Plasma NO(x) correlate negatively with CI in young patients with left-to-right shunt congenital heart diseases.

Molecular cloning, expression, and site-directed mutagenesis of inorganic pyrophosphatase from Thermus thermophilus HB8.

The genomic DNA encoding the inorganic pyrophosphatase from an extremely thermophilic bacterium, Thermus thermophilus HB8 (ATCC27634), was isolated by colony hybridization with a probe designed as a part of gene amplified by the PCR method, which was derived from the partial amino acid sequence of the enzyme. The DNA was cloned into a plasmid vector, pUC118, after digestion with BamHI. The inserted nucleotide fragment was about 1.8 kbp in length and the nucleotide sequence included a 525 bp open reading frame. The deduced amino acid sequence was completely identical with that of the enzyme determined by automated Edman analysis of peptide fragments isolated from digests obtained with Staphylococcus aureus V8 protease and Achromobacter protease I, and also from products obtained on chemical cleavage with cyanogen bromide and 70% formic acid. The subunit of this enzyme is composed of 174 amino acid residues with a calculated molecular weight of 19,084. Then, the gene was overexpressed in Escherichia coli BL21 (DE3) using a plasmid vector, pET15b, system. The recombinant enzyme was fully active, and exhibited higher thermostability than the E. coli enzyme. Amino acid residues located on the surface of the recombinant enzyme were determined by means of limited proteolysis, and the results revealed that the environment of Lys residues is almost the same as the crystal structure reported previously [Teplyakov, A. et al. (1994) Protein Sci. 3, 1098-1107]. Furthermore, the roles of two tryptophan residues were investigated by site-directed mutagenesis, which indicated that they may be responsible for the structural integrity and thermostability.

Fate of the neopulmonary valve after the arterial switch operation in neonates.

OBJECTIVES: The purpose of this study was to determine the incidence, risk factors, and outcomes of acquired stenosis of the neopulmonary valve after the neonatal arterial switch operation. METHODS: Reviewed were the preoperative and follow-up echocardiograms from 136 of 288 patients undergoing the arterial switch operation for whom adequate studies were available. Pulmonary stenosis was defined as a thickened and doming pulmonary valve and a pressure gradient of 20 mm Hg or more. Transposition of the great arteries was present with intact ventricular septum in 91 patients, with a ventricular septal detect in 39, with an aortic coarctation in 5, and with double-outlet right ventricle in 1 patient. No patient had preoperative valvular abnormalities (i.e., a bicuspid valve). RESULTS: During a median follow-up of 18 months (range <1 to 90 months), 32 patients (24%) had the development of supravalvular pulmonary stenosis, 15 (11%) with associated pulmonary valve stenosis (group I). Kaplan-Meier estimates of freedom from any intervention were 94% (95% confidence interval, 90% to 99%) at 1 year and 79% (95% confidence interval, 64% to 94%) at 5 years. The valve anulus before the arterial switch operation was significantly larger (p < 0.03) in those in whom neopulmonary valve stenosis did not develop (group II) than it was in those in whom it did (group I). At follow-up, the pulmonary valve anulus had decreased significantly in diameter in group I (p < 0.0005) and had remained larger in group II (p = 0.06) compared with normal diameter. Group I patients had the development of significant pulmonary valve hypoplasia (p < 0.03) whereas group II patients continued to have significantly larger valves compared with normal size (p < 0.0001). CONCLUSIONS: Neopulmonary valve stenosis after the arterial switch operation is not uncommon and is associated with growth failure of the valve anulus often associated with supravalvular pulmonary stenosis.

Relation between plasma nitrate and mean pulmonary arterial pressure in ventricular septal defect.

BACKGROUND: Nitric oxide (NO) is known to modulate myocardial contraction and coronary tone, and its inhalation reduces pulmonary vascular resistance in patients with pulmonary hypertension. OBJECTIVES: To evaluate the pathophysiological role of NO in patients with a ventricular septal defect (VSD). PATIENTS: Twenty-nine children with VSD, nine of whom had undergone VSD closure surgery, and 14 patients with Kawasaki disease. The mean age of the VSD patients was 3.1 years (range, 2 months to 9 years). METHODS: Using high performance liquid chromatography, nitrate (a more stable NO oxidation product) was measured in plasma specimens of the patients undergoing cardiac catheterisation. RESULTS: Nitrate concentrations in the pulmonary artery bore a significant relation to mean pulmonary artery pressure, pulmonary to systemic systolic pressure ratio, and pulmonary to systemic flow ratio. CONCLUSIONS: The concentration of nitrate was in proportion to the increment in intravascular or cardiac pressure, indicating that endogenous NO is upregulated as a compensatory homeostatic attempt to reduce pulmonary pressure and blood flow.

Multiple hepatoblastomas associated with trisomy 18 in a 3-year-old girl.

A very rare case of full trisomy 18 associated with multiple hepatoblastomas is reported. The patient also had ventricular septal defect and patent ductus arteriosus, which were repaired at 6 months of age. After the cardiac surgery, she was noted to have an abdominal mass and an elevated serum alpha-fetoprotein level. A partial hepatic lobectomy was performed at 7 months of age, and the resected tumor was diagnosed as a fetal-type hepatoblastoma. At 2 years and 4 months of age, a chest radiography disclosed an elevated left diaphragm, and abdominal ultrasonography demonstrated a tumor in the left hepatic lobe. The resected tumor was also diagnosed as a fetal-type hepatoblastoma. Chromosomal analysis demonstrated that the karyotypes of peripheral blood and hepatic tumor cell obtained on two occasions were both 47,XX, +18. She has no evidence of recurrence at 3 years of age without specific therapy.

Double-outlet right ventricle with intact ventricular septum.

An infant with double-outlet right ventricle (DORV) with intact ventricular septum (VS) is described. Hypoplastic left ventricle and mitral valve atresia were also associated. To the authors' knowledge, this appears to be the first case in which this rare form has been demonstrated with magnetic resonance imaging (MRI). The patient required balloon atrial septostomy (BAS) three times until 4 months of age because of progressive cyanosis. In such a form of DORV, surgical atrial septectomy should be performed instead of repeating BAS because the tendency to functional closure of the restrictive secundum arterial septal defect caused by incompetent foramen ovale seems to be prominent.

Eosinophilia in premature infants: correlation with chronic lung disease.

We attempted to clarify the possible pathophysiological significance of eosinophilia in bronchopulmonary dysplasia (BPD). The subjects studied were 17 premature infants, i.e. seven with respiratory distress syndrome (RDS) followed by bronchopulmonary dysplasia (the BPD group: four with stage IV and three with stage III BPD) and 10 infants without BPD (the non-BPD group), who comprised seven with RDS, two with meconium aspiration syndrome and one with transient tachypnea of the newborn. Peripheral eosinophil counts, the number of nuclei of eosinophils and serum eosinophilic cationic protein (ECP) levels, and ECP and polymorphonuclear leukocyte (PMN) elastase levels of intratracheal aspirates (TA) were determined once a week during the first 4 weeks of life. Peripheral eosinophil counts were higher in infants with BPD than those in the non-BPD group. Hypersegmented nuclei of peripheral eosinophils with more than four nuclei were more frequently present in the infants with BPD. A good correlation was observed between peripheral eosinophil counts and serum ECP levels. ECP levels of the TA in the infants with BPD were significantly elevated. There was a good correlation between ECP and PMN elastase levels of the TA. Lung tissue specimens of two infants of the BPD group, both of whom had patent ductus arteriosus (PDA), were obtained from the lower portion of the left lung when they underwent an operative procedure for PDA at 24 and 25 days of life, respectively. Immunohistochemical staining of eosinophil-derived granular major basic protein (MBP) was performed on the lung tissue specimens. Infiltration of a few MBP-staining eosinophils was observed on the specimens from both infants. Our results suggest that peripheral eosinophils in sick premature infants may be activated and appear to be correlated with the severity of BPD. Further studies will be needed to more clarify the physiological role of eosinophils in premature infants.

Direct transaortic balloon valvuloplasty under cardiopulmonary bypass for neonatal critical aortic stenosis.

A 1-day-old male infant with critical aortic valvular stenosis underwent balloon aortic valvuloplasty (BAV) under echocardiographic guidance during cardiopulmonary bypass. Left ventricular function dramatically improved after BAV. This technique combined with a surgical approach was safe and efficient.

Early changes in plasma brain and atrial natriuretic peptides in premature infants: correlation with pulmonary arterial pressure.

To define the change in plasma natriuretic peptides in newborns, we prospectively studied 10 premature infants. They were followed sequentially during the first week of extrauterine life by two-dimensional and pulsed Doppler echocardiography, and studied for atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). We estimated mean pulmonary arterial pressure (MPAP) and measured blood pressure on days 1, 2, 3, 5, 7, respectively. Plasma ANP levels were 81.7 +/- 11.4 pg/ml on day 1 and 67.9 +/- 6.0 pg/ml on day 7, respectively. Between day 2 and day 7, there was a fall in MPAP, i.e. from 37 +/- 4 mmHg to 22 +/- 2 mmHg (P < 0.01), which was associated with a significant decrease in plasma BNP (41.8 +/- 10.1 pg/ml on day 2 vs. 10.4 +/- 0.9 pg/ml on day 7, P < 0.01). There was a positive correlation between MPAP and plasma BNP level (r = 0.643, P < 0.0001), but there was no correlation between MPAP and plasma ANP level. These data suggest that the pattern of secretion of BNP is different from that of ANP and that BNP levels reflect the changes of pulmonary arterial pressure in the neonatal period in premature infants.

[Progression to the dilated phase of hypertrophic cardiomyopathy in children].

Twenty-three children with hypertrophic cardiomyopathy (HCM) (aged 2 months to 15 years) were followed up for more than four years using thallium myocardial imaging (TMI) and echocardiography. With echocardiography, the left ventricular end-diastolic dimension (LVDd) and fractional shortening (FS) were measured. Perfusion defect (PD) was assessed using TMI. Cardiac catheterization and right ventricular endomyocardial biopsy were performed in 18 patients within one week before or after their TMI. During the follow-up period, two patients showed a marked increase in LVDd and a marked decrease in FS at ages 13 and 16 years, respectively. These two patients were judged to have progressed to the dilated phase. In these two patients, extensive PD was detected in the left ventricular wall on TMI, 15 and 31 months prior to the appearance of the echocardiographic changes, respectively. Right ventricular endomyocardial biopsy at the time of extensive PD revealed marked interstitial fibrosis along with hypertrophy and disarray of myocardial cells. In conclusion, progression to the dilated phase is not a rare event in children with HCM and TMI appears to be a useful tool for early detection of the progression.

[Pharmacokinetic and clinical studies on sulbactam/ampicillin in children].

Laboratory and clinical studies of sulbactam/ampicillin (SBT/ABPC) in children have been carried out, and the following results were obtained. 1. Antibacterial effect MICs of SBT/ABPC were only one-tube less than or similar to those of ABPC against susceptible organisms. Against ABPC-resistant organisms at the inoculum size of 10(8) cells/ml however, SBT/ABPC was superior to ABPC when evaluated in terms of their MIC values. When MICs of SBT/ABPC were compared to those of ABPC against organisms with high beta-lactamase producing activities, it was found that many of ABPC-resistant organisms were much susceptible to SBT/ABPC. 2. Absorption and urinary excretion In 2 cases to which 50 mg/kg and 20 mg/kg SBT/ABPC were respectively given over 30 minutes by drip infusion, peak serum levels were obtained at the end of the drip infusion with peak levels of SBT of 45.5 micrograms/ml, 12.5 micrograms/ml, respectively and those of ABPC of 83.0 micrograms/ml, 22.9 micrograms/ml, respectively. The half-lives of SBT and ABPC were 0.94 hour and 0.98 hour, respectively. The mean urinary excretion rates in the first 6 hours after the end of administration were 84.4% for SBT and 63.1% for ABPC. 3. Clinical results Clinical efficacies were evaluated in 24 cases including 9 cases of pneumonia, 2 cases of upper respiratory infection, 7 cases of urinary tract infection and 1 case each of bronchopneumonia, pyothorax, tonsillitis, streptococcal infection, ++ phlegmon and staphylococcal scalded skin syndrome. Clinical efficacies were excellent or good in 19 cases with an overall efficacy rate of 86.4%. Adverse effect was found in 1 case with nausea and vomiting, and abnormal laboratory test values observed were 2 cases each of eosinophilia, slight elevation of GOT and GPT and elevation of LDH, but they were not serious.

[Clinical and pharmacokinetics evaluation of flomoxef in pediatrics].

Flomoxef (FMOX, 6315-S), a new parenteral oxacephem antibiotic was investigated for its clinical efficacy and pharmacokinetics. The results obtained are summarized below. 1. Twenty-eight patients were treated with 39-152 mg/kg per day of FMOX by intravenous administration. Diagnosis of patients were pneumonia in 15 patients, acute upper respiratory tract infection in 5, acute enterocolitis in 3, urinary tract infection in 2 and cholangitis, suppurative lymphadenitis and suspicious sepsis in 1 patient each. Clinical effect was excellent in 7 cases, good in 8, fair in 5, poor in 2 and 6 cases were excluded because therapy periods were too short and other antibiotics were used together. Efficacy rate was 68% and the rate of bacterial disappearance was 83%. 2. Rash was found in 5 cases and thrombocytosis was found in 1 out of 28 cases. However, no severe adverse reaction was encountered. 3. The peak serum level of FMOX was 51.0 micrograms/ml after 20 mg/kg of drip infusion for 30 minutes and the half-life was 17.2 minutes in alpha-phase and 58.2 minutes in beta-phase.