Michael S. Baylerian

Cardiomegaly due to myocyte hyperplasia in perinatal rats exposed to 200 ppm carbon monoxide

Exposure to carbon monoxide (CO) during the fetal and neonatal period was used to evaluate cardiac ventricular regional weight and myocyte growth response to an increased hemodynamic load. Date-mated Sprague-Dawley rats inhaled 200 ppm CO from day 7 of pregnancy until parturition; another group of pregnant rats inhaled room air. At birth, pups from these two groups were subdivided into four groups: (1) control group (AIR/AIR), that was maintained in room air in utero and post-partum; (2) AIR/CO group, which received CO only after birth; (3) CO/CO group, which received CO exposure in utero and post-partum; and (4) CO/AIR group, which received CO exposure in utero but was maintained in room air post-partum. Rats were removed from litters at selected intervals from birth to 28 days of age and used to determine regional ventricular weights and ventricular dry weights, and to obtain isolated myocyte preparations for measurement of cell size characteristics and allow calculation of cell numbers. Compared with AIR raised control animals, right ventricular weight was increased in animals exposed to CO during the fetal period. Post-natal CO exposure caused an increase in left ventricular weight. Heart weight to body weight ratio of animals exposed to CO post-natally only (AIR/CO) gradually increased to reach that of the CO/CO group by 12 days of age, while animals exposed to AIR post-natally following fetal CO exposure gradually decreased their heart weight to body weight ratio toward that of the control animals by 28 days of age. Binucleated cells first appeared at 4 days of age in all groups. Myocyte volume was similar in both groups at birth and increased from six through 28 days of age. Left ventricle plus septum and right ventricle cell volumes of the CO/CO group were smaller than the controls at 28 days of age in spite of heavier wet and dry weights in the CO-exposed rats. At birth, the CO-exposed animals had more myocytes in the RV compared to AIR-exposed controls. Carbon monoxide exposure after birth resulted in left ventricular myocyte hyperplasia. The results of this study indicate that increased hemodynamic load due to CO exposure during the fetal period results in cardiomegaly because of increased myocyte hyperplasia. This cellular response is sustained through the early neonatal period in animals exposed to CO both in utero and post-partum.

Temporary and lasting cardiac effects of pre- and postnatal exposure to carbon monoxide

Abstract Pregnant female rats inhaled 200 ppm CO for the last 18 days of gestation, and a portion of their young inhaled 500 ppm CO for an additional 29 days after birth. Body weight (BW) and litter size of the CO-exposed newborn were significantly lower than those of unexposed controls. Heart weight (HW) of the exposed newborn was significantly larger than the controls, as was also HW BW ratio. Hemoglobin concentration, hematocrit, and red blood cell count were significantly lower in the exposed newborn than in controls In young rats exposed to CO both pre- and postnatally, HW BW ratio was more than twice as large as that of unexposed controls at 14, 21, and 29 days after birth (statistically significant), while in young rats exposed only as fetuses HW BW ratio was only slightly higher than in controls at the same time intervals. The remaining rats in all three groups were allowed to mature to 105 days of age in room air, at which time they too were sacrificed. HW BW ratio of the pre- and postnatally exposed rats was significantly elevated above the same parameters for the controls and rats exposed to CO as fetuses only. The HW BW ratio of the latter two groups did not differ significantly from one another. Analysis of HW data using multiple exponential regression equations relating HW to BW, showed HW of the rats exposed to CO both pre- and postnatally to be 22% larger than predicted. The results suggest that fetal cardiomegaly induced by CO is entirely reversible, while neonatal exposure produces long-standing changes in HW.

Radiometric assays of N-acetylglucosaminylphosphotransferase and α-N-acetylglucosaminyl phosphodiesterase with substrates labeled in the glucosamine moiety

The assay of fibroblast and leukocyte-N-acetylglucosaminylphosphotransferase with alpha-methylmannoside acceptor and commercially available UDP-[3H or 14C]N-acetylglucosamine donor was modified to yield low background and consequently high sensitivity and reliability comparable to those obtained with the synthetically made [beta-32P]UDP-N-acetylglucosamine donor. This was achieved by an additional elution step that removed free [3H or 14C]N-acetylglucosamine which appeared to be the breakdown product responsible for the high background. In addition, the [3H or 14C]N-acetylglucosamine-1-phospho-6-alpha-methylmannoside product of the transfer reaction was then isolated and, following desalting, could serve as a substrate for the assay of alpha-N-acetylglucosaminyl phosphodiesterase. Cell preparations of patients with I-cell disease and pseudo-Hurler polydystrophy demonstrated severe to moderate deficiency of transferase activity and normal phosphodiesterase activity toward the respective substrates labeled with 3H or 14C in the glucosamine moiety.

Thermal activation of hexosaminidase A in a genetic compound with Tay-Sachs disease

Increase in total hexosaminidase activity has been observed during heat treatment of serum and leukocyte specimens from a 1-year-old boy with cherry-red spot and severe and progressive mental and motor deterioration. The activity increased 40% in the first 40–70 min of incubation at 50°C and pH 4.3, but declined thereafter and was only slightly above the initial activity in the final 2–3 h of incubation. Heat treatment of specimens from family members revealed very reduced rates of inactivation of hexosaminidase in the probands father and some paternal relatives, whereas those of the mother and some maternal relatives were indistinguishable from those of Tay-Sachs carriers. Mixing experiments with enzyme preparations from the proband, normal controls and patients with Tay-Sachs disease resulted in additive values and did not support the possibility of inhibitor- or activator-related defect. Fractionation of heat-treated samples by ion exchange chromatography and electrophoresis, as well as examination of the separated fractions for their thermostability, have shown that hexosaminidase A is the activated component and hexosaminidases B, I1 and I2 are not affected. These findings suggest that the patient is a genetic compound and the apparent thermal activation is probably due to formation of hexosaminidase A from altered α-subunits produced by the paternal mutant α-allele and β-subunits produced by the normal β-alleles.

Km defect in neuraminidase of dysmorphic type sialidosis with and without β-galactosidase deficiency

Kinetic studies of 4-methylumbelliferyl neuraminidase activity were carried out in cultured skin fibroblasts from patients with various disorders of neuraminidase deficiency. Cell extracts from two patients with dysmorphic type sialidosis of infantile onset, with isolated deficiency of neuraminidase activity, and three patients with dysmorphic type sialidosis of juvenile onset, with combined deficiency of neuraminidase and beta-galactosidase activities, demonstrated 7-12 times higher apparent Km values than those of normal controls (1.0-1.5 mmol/l as compared with 0.12-0.15 mmol/l). The apparent Ki values for N-acetylneuraminic acid and colominic acid were also increased in the dysmorphic type (7-15 and 7-11 times the normal values, respectively). In contrast, in the normomorphic type, normal apparent Km and Ki values were found for 4-methylumbelliferyl neuraminidase activity in fibroblasts from one patient with isolated neuraminidase deficiency and two patients with combined deficiency of neuraminidase and beta-galactosidase. The altered kinetics in the dysmorphic cases indicates a primary defect in neuraminidase with a secondary deficiency of beta-galactosidase in patients with combined deficiency. It is not clear if the primary defect in the normomorphic cases involves a defect in neuraminidase other than a Km defect or if neuraminidase or both neuraminidase and beta-galactosidase deficiencies are secondary to another defect as yet undetermined.

A study of heart and blood of rodents inhaling diesel engine exhaust particulates

Abstract The in vivo effects of inhalation of diesel engine exhaust (DEE) were evaluated in 128 rats and 146 guinea pigs. They were exposed in special chambers to three different dose levels of DEE particulates; 250, 750, and 1500 μg/m 3 , for 20 hr/day and 5.5 days/week. Rats were sacrificed after 13, 16.7, 25.7, 42, 52, and 78 weeks exposure, while guinea pigs were sacrificed after 6, 13, 17, 26, 42, 52, and 78 weeks exposure. Each group of each species was compared to its own age-matched control group. Morphometric analysis of the heart revealed no significant alterations in mass which could be assigned to inhalation of DEE at any dosage level or duration of exposure in either species. This included an assessment of the relative wet weights of the right ventricle, left ventricle, combined ventricles, combined atria, and ratio of right to left ventricle weights. Likewise, hematology was not changed in either species at any dosage level or duration of exposure by inhalation of DEE.

I-CELL DISEASE AND PSEUDO-HURLER POLYDYSTROPHY; RADIOMETRIC ASSAYS OF N-ACETYLGLUCOSAMINYLPHOSPHOTRANSFERASE AND -N-ACETYLGLUCOSAMINYL PHOSPHODIESTERASE WITH COMMERCIALLY AVAILABLE SUBSTRATES

UDP-N-acetylglucosamine (UDP-GlcNAc) is the donor of N-acetylglucosaminyl-1-phosphate (GlcNAcP) in the reaction catalyzed by GlcNAcP transferase, the enzyme deficient in patients with I-cell disease (ICD) and pseudo-Hurler polydystrophy (PHPD). The use of commercially available UDP-[3H or 14C]GlcNAc rather than the synthetically made [β-32P]UDP-GlcNAc was inadequate because of high background. We have overcome this in the assay of GlcNAcP trasferase with α-methylmannoside acceptor by removal of free [3H or 14C]GlcNAc which appeared to be the major breakdown product. In addition, the α-methylmannose-6-phospho-1-[3H or 14C]GlcNAc product of the transfer reaction was then isolated and following desalting could be used as a substrate for the assay of αGlcNAc phosphodiesterase. Using these relatively simple methods, deficiency of GlcNAcP transferase activity could be demonstrated in fibroblasts from patients with the classical forms of ICD (n=4; less than 4% of control activity) and PHPD (n=4; 3-33% of control activity). αGlcNAc phosphodiesterase activity was within the normal range. In contrast, in three related adult patients with what appears to be a very mild form of PHPD, both activities were normal and utilization of natural lysosomal enzyme acceptor (β-glucuronidase) was required to reveal deficiency of GlcNAcP transferase activity.

CLEAVAGE EN BLOC OF N-ACETYLGLUCOSAMINE-6-SULFATE AND N-ACETYLGALACTOSAMINE-6-SULFATE BY HEXOSAMINIDASE A: DEFICIENCY OF BOTH ACTIVITIES IN TAY-SACHS DISEASE

Current assays of hexosaminidase A (Hex A) are either indirect (heat-inactivation method) or complex (GM2 ganglioside substrate). The finding in Sanfilippo syndrome type D (N-acetylglucosamine-6-sulfatase deficiency) that Hex A can bypass the blockage in keratan sulfate degradation by cleavage en bloc of β-N-acetyl-glucosamine-6-sulfate, led to the present approach. 4-methyl-umbelliferyl derivatives of β-N-acetylglucosamine-6-sulfate (MUβG1cNAc-6-S) and β-N-acetylgalactosamine-6-sulfate (MUβGa1NAc-6-S) were prepared from the commonly used unsulfated derivatives. Sera (n=5), leukocytes (n=3) and fibroblasts (n=2) from patients with Tay-Sachs disease (TSD) demonstrated markedly deficient activities toward both sulfated substrates, 2-4% of control activities (n=12), and samples from obligate heterozygotes for TSD (n=4) had intermediate activity values, 43-62% of control activities. Fibroblasts (n=2) from patients with Morquio syndrome type A (N-acetylgalactosamine-6-sulfatase deficiency) had normal activities toward both sulfated substrates but the characteristic urinary excretion of chondroitin sulfate in this disease, indicates that there is no analogy with Sanfilippo syndrome type D and Hex A is incapable of cleaving en bloc βGa1NAc-6-S from the natural substrate. In contrast to natural substrates, the synthetic sulfated substrates are both specific for Hex A and thus, can provide direct and simple methods for diagnosis as well as for antenatal and carrier detection of TSD.