Long-Term stability of 6-hydroxymelatonin sulfate in 24-h urine samples stored at -20 degrees C.

The purpose of the present feasibility study was to determine whether the concentration of 6-hydroxymelatonin sulfate (6-OHMS) remains stable in urine samples stored over at least 15 yr. To test this, 117 twenty-four-hour urine samples were analyzed, which were obtained from healthy children ages 8 to 9 yr within the periods of 1985-1987,1991-1993, and 1997-1999. 6-OHMS concentrations were determined by enzyme-linked immunosorbent assay. The statistical analyses clearly indicate that the concentration of 6-OHMS remains stable for at least 15 yr if the urine is stored at -20 degrees C.

Real-time PCR analysis of the N-acetyltransferase NAT1 allele *3, *4, *10, *11, *14 and *17 polymorphism in squamous cell cancer of head and neck.

Although tobacco smoke has been established as a main risk factor in the development of head and neck squamous cell cancer (HNSCC), genetic polymorphisms of xenobiotic metabolizing enzymes are supposed to modulate an individual's susceptibility to smoking-related HNSCC. N-acetyltransferase (NAT) 1 gene is known to be polymorphic and its protein product is implicated in the activation and detoxification of carcinogens, such as aromatic amines, present in tobacco smoke. We developed a rapid and reproducible LightCycler-assisted real-time polymerase chain reaction (PCR) for NAT1 genotyping, which allowed the parallel differentiation of NAT1*3, *4, *10 and *11 alleles and separately of NAT1*14 and *17 alleles within 60 min without the need for further post-PCR processing. In order to investigate the role of the NAT1 gene polymorphism as a risk-modifying factor in HNSCC, we tested for the presence of NAT1*3, *4, *10, *11, *14 and *17 alleles in a case-control study of 291 HNSCC patients and 300 healthy controls of Caucasian origin. Our findings suggest that in Caucasians, the risk of HNSCC is not associated with NAT1 polymorphism. The overall distribution of NAT1 allele frequencies was not significantly different among cases and controls. The presence of the fast acetylator NAT1*10 and NAT1*11 alleles did not significantly increase the risk of HNSCC and no modifying effect of NAT1*10 was observed among smokers. This new approach in NAT1 genotyping substantially increases throughput of sample analysis and, therefore, enhances opportunities to study NAT1 as a risk factor in different cancers in large-scale studies.

Pathological excretion patterns of urinary proteins in miners highly exposed to dinitrotoluene.

A cohort of 161 underground miners who had been highly exposed to dinitrotoluene (DNT) in the copper-mining industry of the former German Democratic Republic was reinvestigated for signs of subclinical renal damage. The study included a screening of urinary proteins excreted by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and quantitations of the specific urinary proteins alpha 1-microglobulin and glutathione-S-transferase alpha (GST alpha) as biomarkers for damage of the proximal tubule and glutathione-S-transferase pi (GST pi) for damage of the distal tubule. The exposures were categorized semiquantitatively (low, medium, high, and very high), according to the type and duration of professional contact with DNT. A straight dose-dependence of pathological protein excretion patterns with the semiquantitative ranking of DNT exposure was seen. Most of the previously reported cancer cases of the urinary tract, especially those in the higher exposed groups, were confined to pathological urinary protein excretion patterns. The damage from DNT was directed toward the tubular system. In many cases, the appearance of Tamm-Horsfall protein, a 105-kD protein marker, was noted. Data on the biomarkers alpha 1-microglobulin, GST alpha, and GST pi consistently demonstrated a dose-dependent increase in tubular damage, which confirmed the results of screening by SDS-PAGE and clearly indicated a nephrotoxic effect of DNT under the given conditions of exposure. Within the cluster of cancer patients observed among the DNT-exposed workers, only in exceptional cases were normal biomarker excretions found.

Association of CYP1B1 codon 432 mutant allele in head and neck squamous cell cancer is reflected by somatic mutations of p53 in tumor tissue.

Tobacco use is causally associated with head and neck squamous cell cancer (HNSCC). Here, we present the results of a case-control study that investigated the effects that the genetic variants of the cytochrome (CYP)1A1, CYP1B1, glutathione-S-transferase (GST)M1, GSTT1, and GSTP1 genes have on modifying the risk of smoking-related HNSCC. Allelisms of the CYP1A1, GSTT1, GSTM1, and GSTT1 genes alone were not associated with an increased risk. CYP1B1 codon 432 polymorphism was found to be a putative susceptibility factor in smoking-related HNSCC. The frequency of CYP1B1 polymorphism was significantly higher (P < 0.001) in the group of smoking cases when compared with smoking controls. Additionally, an odds ratio (OR) of 4.53 (2.62-7.98) was discovered when investigating smoking and nonsmoking cases for the susceptible genotype CYP1B1*2/*2, when compared with the presence of the genotype wild type. In combination with polymorphic variants of the GST genes, a synergistic-effect OR was observed. The calculated OR for the combined genotype CYP1B1*2/*2 and GSTM1*2/*2 was 12.8 (4.09-49.7). The calculated OR for the combined genotype was 13.4 (2.92-97.7) for CYP1B1*2/*2 and GSTT1*2/*2, and 24.1 (9.36-70.5) for the combination of CYP1B1*2/*2 and GSTT1-expressors. The impact of the polymorphic variants of the CYP1B1 gene on HNSCC risk is reflected by the strong association with the frequency of somatic mutations of the p53 gene. Smokers with susceptible genotype CYP1B1*2/*2 were 20 times more likely to show evidence of p53 mutations than were those with CYP1B1 wild type. Combined genotype analysis of CYP1B1 and GSTM1 or GSTT1 revealed interactive effects on the occurrence of p53 gene mutations. The results of the present study indicate that polymorphic variants of CYP1B1 relate significantly to the individual susceptibility of smokers to HNSCC.

Contrast thresholds and fixation disparity during 5-Hz sinusoidal single- and dual-axis (vertical and lateral) whole-body vibration.

The present study assumed that whole-body vibration, transmitted through the seat, impairs spatial retinal resolution and oculomotor alignment parallel to the vibration axis. More specifically, that the decrement increases gradually from single-axis lateral via single-axis vertical and dual-axis linear to dual-axis circular motion. Twenty participants (19-26 years of age) with good vision volunteered for the experiment where in three sessions one of the following three conditions, contrast threshold, nonius bias or fixation disparity, for vertically and horizontally oriented test patterns was determined during five experimental conditions. The latter comprised a control (a(z) = a(y) = 0) and four conditions where 5-Hz sinusoidal motion of 1.2 ms(-2) rms were applied separately, either in the vertical or in the lateral direction, or simultaneously in both directions, once without and once with a phase shift of 90 degrees, thus causing dual-axis linear or circular motion. Contrast thresholds for horizontal gratings and the variability of vertical fixation disparity increased significantly whenever the participants were exposed to vertical motion (alone or combined with lateral motion). These effects may result in an increased difficulty in properly recognizing characters and graphic patterns containing horizontal lines and in the development of asthenopic complaints.

Fixation disparity and nonius bias.

Fixation disparity, i.e. the vergence error within Panum's area, can be measured psychophysically with two nonius (vernier) lines that are presented dichoptically, i.e. one to each eye. The observer adjusts these nonius lines to subjective alignment; the resulting physical nonius offset indicates the amount of fixation disparity. The present experiments investigate the relation between fixation disparity and the nonius bias, which is the physical offset of the nonius lines that is adjusted by the observer in order to perceive them as aligned when both nonius lines are presented to both eyes (binocular nonius bias) or both to the left or both to the right eye (monocular nonius bias). It was found that (1) the fixation disparity is correlated with the binocular nonius bias in the horizontal and vertical meridian and (2) the binocular nonius bias can be predicted from the average of the right eye and left eye monocular nonius bias. To remove the influence of the nonius bias on measured fixation disparity it is possible to calculate the fixation disparity relative to the individual binocular nonius bias, rather than to the physical coincidence of the nonius lines. This procedure tends to increase the correlation between fixation disparity and the tonic resting position of vergence. We discuss the clinical relevance of the dichoptic nonius method for measuring fixation disparity and its limitations as compared to physical recordings of eye position.

The significance of lateral whole-body vibrations related to separately and simultaneously applied vertical motions. A validation study of ISO 2631.

Sensitivity of lateral motions relative to vertical motions were determined and compared to predictions provided by ISO 2631. Two experiments were executed where lateral and vertical motions were applied consecutively or simultaneously and where the magnitude of a single- or dual-axis test signal was adjusted until it was judged as equivalent to a preceding single-axis reference motion of the same frequency. Experiment 1: References consisted of vertical sinusoidal motions presented with 1.6-12.5 Hz and weighted accelerations of a(zw) = 0.3, 0.6 and 1.2 m s(-2) r.m.s., single-axis test signals were lateral motions of the same frequency. 26 subjects (15 men, 11 women, 20-56 yr) participated in the experiments. Accelerations adjusted for lateral vibrations above 1.6 Hz were considerably lower than predicted suggesting that the weighting factors provided in ISO 2631 are incorrect. Experiment 2: References consisted of single-axis vertical or lateral sinusoidal motions presented with 1.6-12.5 Hz and a weighted acceleration of a(zw) = 1.25 m s(-2) r.m.s. The dual-axis test signals consisted of a constant fraction of the reference acceleration (10, 25, 50, 75, 90%) and a perpendicularly oriented adjustable component. 31 subjects (15 men, 16 women, 19-51 yr) participated in the experiments. Both experiments revealed that ISO 2631 is qualitatively valid, the weighting of lateral motions above 1.6 Hz, however, should be increased in order to meet the actual sensitivity particularly in case of multi-axis vibrations.

Subtilisin BPN' variants: increased hydrolytic activity on surface-bound substrates via decreased surface activity.

Site-directed mutagenesis and random mutagenesis were used to produce variants of subtilisin BPN' (Bacillus amyloliquefaciens) protease with variable surface adsorption properties. Protease adsorption and peptide hydrolysis rate were measured for these variants using a model substrate consisting of a peptide covalently bound to a surface. While most variants adsorb at a level very similar to that of native BPN', several variants were identified which adsorb either more or less. For surface-bound substrates we report a linear dependence between the concentration of adsorbed protease enzyme and substrate hydrolysis, similar to the linear dependence between enzyme solution concentration and hydrolysis of soluble substrates. On the basis of this knowledge we hypothesized that variants designed to adsorb at a higher level on a surface-bound peptide substrate would hydrolyze that surface-bound substrate faster. Contrary to our original expectations, the variants that adsorb more on the covalently bound peptide surface hydrolyze this substrate slower. In addition, variants of BPN' which adsorb at a lower level than native BPN' hydrolyze the surface-bound substrate faster. Enzyme adsorption and the subsequent peptide hydrolysis are altered by substituting amino acids that modify the surface charge or hydrophobicity of the native enzyme. This effect is most dramatic when the changes were made at surface-exposed sites around the binding pocket/active site of the enzyme. One mechanism that is consistent with the data is based on the relationship between the level of adsorption and the enzyme's affinity for the surface. In this mechanism weakly adsorbed enzymes are postulated to move more rapidly from site to site on the surface, thereby increasing substrate hydrolysis.

Enzyme behavior at surfaces. Site-specific variants of subtilisin BPN' with enhanced surface stability.

Enzyme adsorption and inactivation at the solid/liquid interface for subtilisin BPN' show a strong dependence on the nature of the solid surface. Adsorption of BPN' at the solid/liquid interface is considerably greater for a hydrophobic surface than for a hydrophilic one. Likewise, the rate of inactivation of the wild-type BPN' is over five times greater when equilibrated with a hydrophobic surface than with a hydrophilic surface. The rate data from these enzyme inactivation experiments performed at 50 degrees C are best fit by a second-order kinetic equation, suggesting a bimolecular pathway to inactivation. The role of increased surface adsorption on this bimolecular inactivation is discussed in terms of two different mechanisms. Several site-specific variants of subtilisin BPN' have been made in an attempt to alter the surface-inactivation of the wild-type enzyme. The extent of adsorption on the model surfaces is significantly lowered by certain lysine to phenylalanine changes in BPN'. Consequently, the surface autolytic stability shows a 4-fold improvement. The change in surface autolytic stability is achieved even though the basic kinetic parameters (kcat and KM) of the variant enzymes are not significantly different on a soluble substrate. The results provide insights into the use of mutagenesis to probe the mechanism of protein interactions with surfaces.

Echocardiographic proof of pulmonary hypertension with irreversible increased resistance in the pulmonary circulation as a complication after placement of a ventriculo-atrial shunt for internal hydrocephalus.

Increased resistance in the pulmonary vessels in children with ventriculo-atrial shunts is a rare and often unrecognized permanent complication. We report 2 children in whom this diagnosis was detected by two-dimensional echocardiography. The first patient received a ventriculo-atrial shunt at age 9 days for congenital internal hydrocephalus. At 17 months it had to be replaced because of infection of the efferent catheter limb. At 22 months at a routine follow-up the echocardiographic diagnosis of pulmonary hypertension was made. Invasive studies confirmed the presence of irreversible increased resistance in the pulmonary circulation. The second patient received a ventriculo-atrial shunt at age 13 months because of a cerebral cyst. After repeated catheter infections, at 28 months a ventriculo-peritoneal shunt was placed. At age 4 years the diagnosis of pulmonary hypertension was made by routine echocardiography. This finding was confirmed by invasive studies. The left pulmonary artery was completely occluded. Both patients had developed microemboli, caused or aggravated by catheter sepsis, in the second case probably through contiguous clot growth up to complete occlusion of the left pulmonary artery. Therapeutic measures seemed not to be indicated. Two-dimensional echocardiography proved to be a reliable method for diagnosing increased resistance and pulmonary hypertension. We recommend routine echocardiography for follow-up in all children with ventriculo-atrial shunts.

Dynamic exercise echocardiography of the left ventricle in physically trained children compared to untrained healthy children.

To evaluate the effect of physical training on left ventricular function, we investigated 52 children, aged 7 to 14 years, who had trained for endurance sports over a period of at least 1 year. The children (24 boys and 28 girls) were investigated echocardiographically during supine exercise on a bicycle at increasing work loads of 6, 9, 12 and 15 kpm/min/kg body weight. The parameters of left ventricular function (specifically fractional shortening and the velocity of circumferential fiber shortening; both these parameters corrected for heart rate) were evaluated before, during and after the test. The values obtained were compared to those in untrained children investigated in a previous study. Fractional shortening in trained children rose from 37 +/- 5% to 54 +/- 5%, and in untrained children from 37 +/- 4% to 46 +/- 4%. The velocity of fiber shortening in trained children rose from 1.27 to 3.15 circ/sec while in untrained children it increased from 1.25 to 2.53 circ/sec. Left ventricular contraction, therefore was significantly greater in trained than in untrained children during exercise. While untrained children increased their cardiac output in the first minutes of exercise mainly by elevating their heart rate, trained children increased simultaneously the stroke volume. There were no significant differences between boys and girls, or between stages of maturity. Thus, physical training causes quick adaptations of left ventricular function to exercise in children of all ages.

[Pulmonary sequestration in infants: diagnosis and therapy exemplified by a newborn infant]. / Lungensequester im Säuglingsalter: Diagnostik und Therapie am Beispiel eines Neugeborenen.

Infants with intra- or extralobular lung-sequestrations normally suffer from chronic infections. In our case an infant is described who had a triangular area in chest X-rays behind the heart. Lung scintigraphy and computer-tomography could not commit the diagnosis. Only invasive angiography showed the sequestration. Although without symptoms the baby was operated preventively. Postoperative course was without complications.

Dynamic exercise echocardiography in children with congenital heart disease affecting the left heart.

Thirty-five children (male 22, female 13) with congenital heart disease resulting in volume and pressure overload of the left ventricle were investigated echocardiographically during supine bicycle exercise. The children had to follow a test-protocol with increasing workload. Left ventricular function parameters were measured from M-mode-echocardiography, electrocardiography and phonocardiograms before, during and after exercise and were expressed as fractional shortening (FS), velocity of circumferential fiber shortening (VcF) and the frequency corrected parameters: FScorr. = FS X 100/HR and VcFcorr. = VcF X 100/HR (HR = heart rate). The data of this group of children under study were compared to those obtained from 140 healthy children examined under similar conditions. In children with pressure overload, values for fractional shortening, velocity of circumferential fiber shortening and the frequency corrected parameters were significantly higher than in normals throughout exercise testing. In some children with moderate to severe aortic stenosis or coarctation the frequency corrected parameters showed a decrease at higher exercise levels instead of an increase as seen in the majority of cases. In these cases cardiac output was increased by an abnormal rise in heart rate. This was considered as a diminished left ventricular reserve. After aortic valve replacement in two cases with aortic stenosis, parameters of left ventricular function were still elevated at rest and during exercise testing. In two children with hypertrophic cardiomyopathy the almost maximally elevated rest values did not change during exercise. In children with mild volume overload (small ventricular septal defect or aortic incompetence) the left ventricular function parameters were within the normal range or slightly below.

Echocardiographic evaluation of left ventricular reserve in normal children during supine bicycle exercise.

To evaluate left ventricular reserve in normal children 127 healthy boys and girls (71 males, 56 females) were investigated with echocardiography during supine bicycle exercise at levels of 6, 9, 12 and 15 kpm/min/kg body weight. Left ventricular function parameters were obtained from the M-mode echocardiogram, electrocardiogram and phonocardiogram. The children were separated into three groups according to body surface area: group I less than 1.1 m2, group II 1.1-1.4 m2, and group III greater than 1.4 m2. Fractional shortening (FS) of the left ventricle rose during exercise from 37 +/- 4 to 46 +/- 4% with no significant differences between the three groups. Velocity of circumferential fiber shortening (Vcf) in group I augmented from 1.3 to 2.25 circ./sec, in group II from 1.29 to 2.60 circ./sec, and in group III from 1.17 to 2.74 circ./sec (P = 0.01). In the recovery period heart rate (HR) and blood pressure normalized earlier than did fractional shortening and velocity of circumferential fiber shortening. This could be expressed best by the ratios FS/HR and Vcf/HR. There were no significant differences between sexes, although there was a tendency to higher blood pressure and heart rate, a greater increase in velocity of circumferential fiber shortening and a smaller increase in fractional shortening in girls. These data indicate that girls increased cardiac output during exercise more by increasing their heart rate than did boys.