The amplification refractory mutation system (ARMS): a rapid and direct prenatal diagnostic technique for beta-thalassaemia in Singapore

beta-Thalassaemia major patients have chronic anaemia and since 3-4 per cent of Singaporeans carry the beta-gene, prenatal diagnosis is essential. We evaluated the amplification refractory mutation system (ARMS) technique as a routine test for prenatal diagnosis of beta-major. Six mutations along the beta-gene were studied--41-42 (-TCTT), IVSII #654 (C-T), 17 beta (A-T), -28 TATA (A-G), IVSI #5 (G-C), and IVSI #1 (G-T). Our results indicate that prenatal diagnosis using these mutations can be offered to 90 per cent (35/39) of our Chinese couples and 54.6 per cent (12/22) of our Malay couples at risk. Confirmation of ARMS results was carried out using allele-specific oligonucleotide hybridization. Prenatal diagnosis using ARMS was successfully carried out in nine cases which included a set of triplets and twins. The triplets were diagnosed with the beta-trait carrying the 41-42 mutation. The couple with twins possessed the #654 mutation and one twin was diagnosed with the beta-trait and the other with #654 homozygosity. Genomic sequencing of the undefined mutations in the Chinese couples revealed rarer mutations at -29 and an ATG-AGG base substitution at the initiation codon for translation. In the Malay couples, genomic sequencing detected mutations at codon 15 (TGG-TAG) and codon 26 (GAG-AAG). We conclude that ARMS with its direct detection of amplified products by gel electrophoresis provides an accurate, rapid, and simpler method for our beta-thalassaemia prenatal diagnosis programme in Singapore.     

Quantitative renal scintigraphic determination of effective renal plasma flow in dogs with normal and abnormal renal function, using 99mTc-mercaptoacetyltriglycine

Effective renal plasma flow (ERPF) was evaluated, using the measurement of p-aminohippurate clearance (CLPAH) and quantitative renal scintigraphy (QRS) with 99mTc-mercaptoacetyltriglycine (99mTc-MAG3). The CLPAH and QRS determinations were made in 6 dogs: 2 determinations for each dog before, and 1 determination after induction of renal failure by administration of amphotericin B. Least-squares regression analysis was used to derive an equation to estimate ERPF from QRS data. The results indicated that QRS, using 99mTc-MAG3, correlated reasonably well (r = 0.82, P < 0.001) with ERPF determined from the CLPAH value. The right kidney contributed 53.3% of global ERPF (P = 0.002). Hepatobiliary excretion of 99mTc-MAG3 was variable within each dog. There was not a consistent pattern with respect to time or renal function. All dogs had nausea or emesis, or both, after IV administration of 99mTc-MAG3. The QRS method with 99mTc-MAG3 provides an adequate means to estimate ERPF in healthy dogs and dogs with renal failure.     

Long-term erythropoietin expression in rodents and non-human primates following intramuscular injection of a replication-defective adenoviral vector

Erythropoietin (Epo)-responsive anemia is a debilitating complication of chronic renal failure and human immunodeficiency virus (HIV) infection that effects more than 150,000 Americans. Patients with Epo-responsive anemias are currently treated with repeated injections of recombinant human Epo. In the studies described in this report, we have examined the safety and efficacy of using a single intramuscular (i.m.) injection of replication-defective adenoviral vectors (RDAd) encoding Epo for the treatment of Epo-responsive anemias in both mice and non-human primates. Our results demonstrate that there is a threshold dose of virus (2.5-8 x 10(7) pfu/gram of body weight) which is required to obtain long-term Epo expression and polycythemia in both species. A single i.m. injection of mice with 10(9) pfu of an RDAd encoding murine Epo (AdmEpo) resulted in elevations in hematocrits from control values of 49 +/- 0.9% to treated values of 81 +/- 3%, which were stable for more than 1 year. Similarly, a single i.m. injection of a monkey with 4 x 10(11) pfu of an RDAd-encoding simian Epo (AdsEpo) resulted in elevations of hematocrits from control levels of 40% to treated levels of > or =70%, which were stable for 84 days. Intramuscular injection of monkeys with AdsEpo appeared to be safe in that we did not detect abnormalities in chest X-rays, serum chemistries, hematologic, or clotting profiles (apart from elevated hematocrits) or organ histologies during the 84-day time course of the experiment. Taken together, these results suggest the feasibility of using i.m. injection of RDAd for the treatment of Epo-responsive anemias in humans.     

Solution scattering structural analysis of the 70 S Escherichia coli ribosome by contrast variation. I. Invariants and validation of electron microscopy models

Solutions of selectively deuterated 70 S Escherichia coli ribosomes and of free 30 S and 50 S subunits were studied by neutron scattering using contrast variation. The integrity of the partially deuterated particles was controlled by parallel X-ray measurements. Integral parameters of the entire ribosome, of its subunits and of the protein and rRNA moieties were evaluated. The data allow an experimental validation of the two most recent electron microscopy reconstructions of the 70 S ribosome presented by the groups of J. Frank (Albany) and of M. van Heel & R. Brimacombe (Berlin). For each reconstruction, integral parameters and theoretical scattering curves from the 70 S and its subunits were calculated and compared with the experimental data. Although neither of the two models yields a comprehensive agreement with the experimental data, Frank's model provides a better fit. For the 50 S subunit of van Heel & Brimacombe's model the fit with the experimental data improves significantly when the internal channels and tunnels are filled up. The poorer fit of the latter model is thus caused by its "sponge"-like structure which may partly be due to an enhancement of high frequency contributions in some of the steps of the three-dimensional image reconstruction. It seems therefore unlikely that the ribosome has a "sponge"-like structure with a pronounced network of channels.     

Correlation between secretagogue-induced Ca2+ influx, intracellular Ca2+ levels and secretion of catecholamines in cultured adrenal chromaffin cells

BACKGROUND: Complex branched muscle fibers are frequently observed in the muscles of mdx mutant mice and/or in damaged muscles. To investigate whether the complex branched fibers were present in the compensatory hypertrophied muscles of rats, we examined the morphological changes in these muscles. METHODS: We examined the hypertrophied plantaris (PLA) muscle of the Wistar male rats, prepared by surgical ablation of synergistic muscles. The muscle was examined using three-dimensional analysis with scanning electron microscopy, immunohistochemical detection of proliferating cells using 5-bromo-2'-deoxyuridine (BrdU) and histological and histochemical characterization. Studies were performed at 48 hours, 2, 4, 6, 10, and 15 weeks after surgical preparation. RESULTS: The muscle hypertrophy ratio (muscle weight relative to the contralateral intact control side), gradually increased from 2 to 10 weeks, and the peak value (48.6%) occurred at the 10th week. The total number of fibers did not change significantly at any time interval. However, the number of branched muscle fibers increased significantly (P < 0.05) after 6 weeks, and accounted for about 2.5% of the total fibers at the 15th week. Most branched fibers showed complex features resembling the "anastomosing syncytial reticulum" described in myopathic animals. The fibers were observed mainly in the middle and distal portions of the PLA muscle. The proportion and distribution of proliferating cells in the entire PLA muscle corresponded with the distribution of the complex branched fibers. These results were also observed in muscle tissues prepared for histological and histochemical examination. CONCLUSIONS: The presence of a large proportion of complex branched fibers in a limited segment of the compensatory hypertrophied muscle suggests that this hypertrophy model represents a pathological and/or pathophysiological hypertrophy model rather than a normal physiological process.     

Overexpression of human lecithin:cholesterol acyltransferase in cholesterol-fed rabbits: LDL metabolism and HDL metabolism are affected in a gene dose-dependent manner

Lecithin:cholesterol acyltransferase (LCAT) is an enzyme well known for its involvement in the intravascular metabolism of high density lipoproteins; however, its role in the regulation of apolipoprotein (apo) B-containing lipoproteins remains elusive. The present study was designed to investigate the metabolic mechanisms responsible for the differential lipoprotein response observed between cholesterol-fed hLCAT transgenic and control rabbits. 131I-labeled HDL apoA-I and 125I-labeled LDL kinetics were assessed in age- and sex-matched groups of rabbits with high (HE), low (LE), or no hLCAT expression after 6 weeks on a 0.3% cholesterol diet. In HE, the mean total cholesterol concentration on this diet, mg/dl (230 +/- 50), was not significantly different from that of either LE (313 +/- 46) or controls (332 +/- 52) due to the elevated level of HDL-C observed in HE (127 +/- 19), as compared with both LE (100 +/- 33) and controls (31 +/- 4). In contrast, the mean nonHDL-C concentration for HE (103 +/- 33) was much lower than that for either LE (213 +/- 39) or controls (301 +/- 55). FPLC analysis of plasma confirmed that HDL was the predominant lipoprotein class in HE on the cholesterol diet, whereas cholesteryl ester-rich, apoB-containing lipoproteins characterized the plasma of LE and, most notably, of controls. In vivo kinetic experiments demonstrated that the differences in HDL levels noted between the three groups were attributable to distinctive rates of apoA-I catabolism, with the mean fractional catabolic rate (FCR, d-1) of apoA-I slowest in HE (0.282 +/- 0.03), followed by LE (0.340 +/- 0.01) and controls (0.496 +/- 0.04). A similar, but opposite, pattern was observed for nonHDL-C levels and LDL metabolism (h-1), such that HE had the lowest nonHDL-C levels with the fastest rate of clearance (0.131 +/- 0.027), followed by LE (0.057 +/- 0.009) and controls (0.031 +/- 0.001). Strong correlations were noted between LCAT activity and both apoA-I (r= -0.868, P < 0.01) and LDL (r = 0.670, P = 0.06) FCR, indicating that LCAT activity played a major role in the mediation of lipoprotein metabolism. In summary, these data are the first to show that LCAT overexpression can regulate both LDL and HDL metabolism in cholesterol-fed rabbits and provide a potential explanation for the prevention of diet-induced atherosclerosis observed in our previous study.