Receptors for bradykinin (BK) were characterized in primary cultures of beating neonatal rat cardiomyocytes using [3H]BK was radioligand. Degradation studies demonstrated that [3H]BK was stable for at least 2 h when incubated with cardiomyocytes at 2 and 37 degrees C in the presence of bacitracin in combination with captopril or ramiprilat. Without these inhibitors, > 80% of the [3H]BK was degraded within 2 h at 37 degrees C. This indicates that angiotensin-converting enzyme (ACE) is responsible for the main BK-degrading activity in cardiomyocytes. Scatchard plots were linear and gave a Kd of 1.5 +/- 0.8 nmol/l (mean +/- SD, n = 4) and a maximum binding capacity of 55-125 fmol/mg protein. Association and dissociation studies showed that binding of [3H]BK was saturable and reversible. Binding of [3H]BK at 37 degrees C led to internalization of the ligand. Competition studies with B1 and B2 agonists and antagonists were consistent with a B2 subtype of receptor. Addition of BK to beating cardiomyocytes (> 1 nmol/l) at 37 degrees C gave a strong but transient negative chronotropic effect. This response was paralleled by changes in the pulsation amplitude, which indicated a simultaneous negative inotropic effect of BK. These results provide a basis for the hypothesis that ACE inhibition exerts its cardioprotective effect at the level of a population of cardiomyocytes by virtue of kinin receptor-mediated mechanisms.
Magnetic force combined with magnetic nanoparticles recently has shown potential for enhancing nucleic acid delivery. Achieving effective siRNA delivery into primary cultured cells is challenging. We compared the utility of magnetofection with lipofection procedures for siRNA delivery to primary and immortalized mammalian fibroblasts. Transfection efficiency and cell viability were analyzed by flow cytometry and effects of gene knockdown were quantified by real-time PCR. Lipofectamine 2000 and magnetofection achieved high transfection efficiencies comparable to similar gene silencing effects of about 80%; the cytotoxic effect of magnetofection, however, was significantly less. Magnetofection is a reliable and gentle alternative method with low cytotoxicity for siRNA delivery into difficult to transfect cells such as mammalian fibroblasts. These features are especially advantageous for functional end point analyses of gene silencing, e.g., on the metabolite level.
Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) represents a potentially fatal fatty acid beta-oxidation disorder. Newborn screening (NBS) by tandem mass spectrometry (MS/MS) has been implemented worldwide, but is associated with unresolved questions regarding population heterogeneity, burden on healthy carriers, cut-off policies, false-positive and negative rates. In a retrospective case-control study, 333 NBS samples showing borderline acylcarnitine patterns but not reaching recall criteria were genotyped for the two most common mutations (c.985A>G/c.199C>T) and compared with genotypes and acylcarnitines of 333 controls, 68 false-positives, and 34 patients. c.985A>G was more frequently identified in the study group and false-positives compared to controls (1:4.3/1:2.3 vs. 1:42), whereas c.199C>T was found more frequently only within the false-positives (1:23). Biochemical criteria were devised to differentiate homozygous (c.985A>G), compound heterozygous (c.985A>G/c.199C>T), and heterozygous individuals. Four false-negatives were identified because our initial algorithm required an elevation of octanoylcarnitine (C(8)) and three secondary markers in the initial and follow-up sample. The new approach allowed a reduction of false-positives (by defining high cut-offs: 1.4 micromol/l for C(8); 7 for C(8)/C(12)) and false-negatives (by sequencing the ACADM gene of few suspicious samples). Our validation strategy is able to differentiate healthy carriers from patients doubling the positive predictive value (42-->88%) and to target NBS to MCADD-subsets with potentially higher risk of adverse outcome. It remains controversial, if NBS programs should aim at identifying all subsets of all diseases included. Because the natural course of milder variants cannot be assessed by observational studies, our strategy could serve as a general model for evaluation of MS/MS-based NBS.
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