ecNOS mRNA concentrations were analyzed by reverse transcription-PCR (RT-PCR), as described previously, with minor modifications (19).
After each blot was blocked with 50 g/L nonfat dried milk, it was incubated in primary mouse monoclonal antibody specific for human ecNOS (1:2000 dilution).
The pellets were resuspended in homogenization buffer, and ecNOS activity in the preparation was measured by the conversion of [3H]arginine to [3H]citrulline with use a Nitric Oxide Synthase Assay Kit (Calbiochem), according to the manufacturer's instructions.
ecNOS POLYMORPHISMS IN HUMAN UMBILICAL CORDS In the case of the intron 4 VNTR, 9 of 43 individuals had the 4a/4b genotype, and the others had the 4b/4b genotype.
To quantify differences in ecNOS expression according to the genotypes of the intron 4 VNTR and the E298A polymorphism, ecNOS and GAPDH mRNA were reverse-transcribed and amplified in the same tube (multiplex RT-PCR).
We measured ecNOS protein concentrations, according to the genotypes of the intron 4 VNTR and the E298A polymorphism, by Western blotting; the results are summarized in Fig.
The ecNOS enzyme activities in cultured HUVECs, according to the genotypes of the intron 4 VNTR and E298A polymorphism, are summarized in Fig.
CAD is a multifactorial disease that may differ in each race or ethnic group; for example, the prevalence of CAD differs widely among different populations, and the frequencies of ecNOS gene polymorphisms have been reported to vary among ethnic groups (12).
In the present study, the association between CAD and three polymorphisms of the ecNOS gene was investigated.
The E298D polymorphism in exon 7 of the ecNOS gene has been reported to be a strong risk factor for CAD (10), and Wang et al.
A dependency between increased plasma NOx and the polymorphisms of the ecNOS gene is observed in controls with the E298D mutation.
 Nonstandard abbreviations: NOS, nitric oxide synthase; CAD, coronary artery disease; NOx, [NO.sup.-.sub.2] plus [NO.sup.-.sub.3]; ecNOS, constitutive endothelial NOS; and VNTR, variable number of tandem repeats.