Rapid combined genotyping of factor V, prothrombin and methylenetetrahydrofolate reductase single nucleotide polymorphisms using minor groove binding DNA oligonucleotides (MGB probes) and real-time polymerase chain reaction.

Risk factors for cardiovascular diseases and venous thromboembolism involve both acquired and hereditary conditions. Among the latter, mutations in genes coding for coagulation factors (factor V Leiden [Arg506Gly], G20210A in the 3'-untranslated region of factor II ) and variant C677T of the methylenetetrahydrofolate reductase (MTHFR ) are often involved and co-inherited. These three factors were genotyped simultaneously in the same 96-well plate, using a real-time polymerase chain reaction (PCR) Taqman assay and minor groove binding DNA oligonucleotides (MGB probes). While primers and MGB probes matched their corresponding single nucleotide polymorphism (SNP), the real-time MGB program was identical for each target gene. Homozygous wild-type (WT; -/-), heterozygous (+/-) or homozygous (+/+) variants (n = 362) were selected for factor V (n = 115, with -/-, 40; +/-, 40; +/+, 35), factor II (n = 122, with -/-, 60; +/-, 60; +/+, 2), and MTHFR (n = 120, with -/-, 40; +/-, 40; +/+, 40), according to the results of conventional PCR-restriction fragment length polymorphism (PCR-RFLP), but the allelic discrimination was performed blind. Results of the real-time MGB and PCR-RFLP assays were identical. This new assay was easy and fast with high throughput, without risk of molecular carryover, and cost-effective for laboratories utilizing the Taqman or related fluorescence reading methods. These advantages make it particularly suitable for large-scale combined genotyping of several polymorphisms in the routine setting.     

Calcium antagonists inhibit positive chronotropic responses to alpha 1-adrenoceptor activation in rat isolated atria

Positive chronotropic responses of rat isolated atria to phenylephrine were reduced by propranolol (0.3 microM) and the residual response was further depressed by the selective alpha 1-adrenoceptor antagonist prazosin (1 nM) but not yohimbine (10 nM), confirming that a component of the response to phenylephrine was due to activation of alpha 1-adrenoceptors. When beta-adrenoceptors were blocked by propranolol, the positive chronotropic response to phenylephrine was enhanced by increasing the calcium concentration and by the calcium channel activator Bay K 8644 (0.1 microM), whereas the response was decreased by lowering the calcium concentration and by the calcium antagonists verapamil (10 nM), nifedipine (10 nM) and diltiazem (100 nM). In the presence of prazosin, when phenylephrine acts only on beta-adrenoceptors, calcium antagonists had no effect on the response. In rat isolated aortic strips in a calcium-free, high K+ (40 mM) solution, verapamil (10 nM), nifedipine (10 nM) and diltiazem (100 nM) shifted the calcium-induced contraction curves to the right, but prazosin (10 nM) had no effect, indicating that it is not a calcium antagonist. The calcium antagonists in the concentrations stated above had no effect on phenylephrine-induced contractions of rat aortic strips in normal Krebs-Henseleit solution, indicating that they did not block alpha 1-adrenoceptors in these concentrations. Taken together, these data suggest that the positive chronotropic effect of phenylephrine resulting from activation of alpha 1-adrenoceptors involves an increased influx of calcium through channels that are sensitive to organic calcium antagonists.     

An evaluation of the Kodak Ektachem clinical chemistry slide for theophylline

We evaluated the Kodak Ektachem clinical chemistry slide for assay of theophylline. Assay precision and accuracy were acceptable in the therapeutic range although precision was poor at low levels of theophylline. The assay performed well with patients' samples using the Abbott TDx as the reference procedure but, as indicated by the manufacturer, uremic samples gave a positive bias, particularly in the therapeutic range. Finally, the significant bias observed with Quality Control material, probably due to matrix sensitivity, is a possible drawback.     

Immunomodulation of Delayed Hypersensitivity to Methylated Bovine Serum Albumin (MBSA) in Mice Using Subliminal and Normal Sensitization Procedures

Delayed hypersensitivity (DH) was induced in the footpads of mice sensitized to methylated bovine serum albumin (MBSA). The magnitude of this DH response increased with increasing sensitizing concentration of MBSA. Levamisole administered 1 hr prior to MBSA challenge stimulated the DH response and this was optimal using subliminal sensitizing concentrations of antigen. A number of antirheumatic agents, immunomodulators mediator antagonists and antiallergies were subsequently examined using the subliminal sensitizing concentration of MBSA. The same drugs were also evaluated using a normal sensitizing procedure. These studies indicate that the sensitizing concentration of antigen is critical in establishing whether a drug will stimulate or suppress a DH response.     

Yucatan Pig: An Optimal Hairless Model for a True Random Cutaneous Flap

Porcine models have been used extensively for skin flap research because of the established similarity between the cutaneous blood supply of the swine and humans. The Yucatan minipig provides an excellent model for researching the properties of random cutaneous flaps, offering several advantages over other breeds of swine. In this study, a total of 67 random cutaneous dorsal flank flaps measuring 4 × 14 cm were raised on nine Yucatan minipigs. The mean survival length (10.03 ± 1.60 cm) of the nondelayed flaps was greater than others reported in the literature. The well-defined plane between the subcutaneous tissue and the panniculus carnosus facilitated flap elevation consistently above the level of the panniculus carnosus thereby ensuring the creation of a true random cutaneous flap. Furthermore, the hairless nature of the skin, particularly beneficial in studying chemical peels, permits easy visualization and monitoring of any external skin changes. These advantages make the Yucatan minipig a more desirable alternative to other breeds of swine for use in skin flap research.     

Olfaction in essential tremor patients with and without isolated rest tremor.

An olfactory deficit is present in patients with essential tremor (ET), but it is often milder than that in patients with Parkinson's disease (PD). In both, the deficit occurs early in the disease. Isolated rest tremor without other signs of parkinsonism can occur in patients with ET. If the rest tremor in these patients represents a manifestation of ET rather than early PD, we hypothesized that their University of Pennsylvania Smell Identification Test (UPSIT) scores would be similar to those of ET patients without rest tremor. The mean UPSIT score in 13 ET patients with isolated rest tremor did not differ from that of 58 ET patients without rest tremor (29.3 +/- 4.3 vs. 29.4 +/- 6.4; P = 0.69). Several ET patients with rest tremor had UPSIT scores that fell outside of the range that is seen in 95% of patients with PD. These data raise the possibility that some ET patients with isolated rest tremor may not have early PD and that the pathological process that is responsible for their ET is also involving the basal ganglia.