Histamine N-methyltransferase (HNMT) catalyzes a major pathway in histamine metabolism. Levels of HNMT activity in humans are regulated by inheritance. We set out to study the molecular basis for this genetic regulation. Northern blot analysis showed that HNMT is highly expressed in the kidney, so we determined levels of enzyme activity and thermal stability in 127 human renal biopsy samples. DNA was isolated from 12 kidney samples with widely different HNMT phenotypes, and exons of the HNMT gene were amplified with the polymerase chain reaction. In these 12 samples, we observed a C314T transition that resulted in a Thr105Ile change in encoded amino acid, as well as an A939G transition within the 3'-untranslated region. All remaining renal biopsy samples then were genotyped for these two variant sequences. Frequencies of the alleles encoding Thr105 and Ile105 in the 114 samples studied were 0.90 and 0.10, respectively, whereas frequencies for the nucleotide A939 and G alleles were 0.79 and 0.21, respectively. Kidney samples with the allele encoding Ile105 had significantly lower levels of HNMT activity and thermal stability than did those with the allele that encoded Thr105. These observations were confirmed by transient expression in COS-1 cells of constructs that contained all four alleles for these two polymorphisms. COS-1 cells transfected with the Ile105 allele had significantly lower HNMT activity and immunoreactive HNMT protein than did those transfected with the Thr105 allele. These observations will make it possible to test the hypothesis that genetic polymorphisms for HNMT may play a role in the pathophysiology of human disease.
Johne's disease (JD), or paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis, is one of the most widespread and economically important diseases of livestock and wild ruminants worldwide. Control of JD could be accomplished by diagnosis and good animal husbandry, but this is currently not feasible because commercially available diagnostic tests have low sensitivity levels and are incapable of diagnosing prepatent infections. In this study, a highly sensitive and subspecies-specific enzyme-linked immunosorbent assay was developed for the diagnosis of JD by using antigens extracted from the surface of M. avium subsp. paratuberculosis. Nine different chemicals and various intervals of agitation by vortex were evaluated for their ability to extract the surface antigens. Various quantities of surface antigens per well in a 96-well microtiter plate were also tested. The greatest differences in distinguishing between JD-positive and JD-negative serum samples by ethanol vortex enzyme-linked immunosorbent assay (EVELISA) were obtained with surface antigens dislodged from 50 g/well of bacilli treated with 80% ethanol followed by a 30-second interval of agitation by vortex. The diagnostic specificity and sensitivity of the EVELISA were 97.4% and 100%, respectively. EVELISA plates that had been vacuum-sealed and then tested 7 weeks later (the longest interval tested) had diagnostic specificity and sensitivity rates of 96.9 and 100%, respectively. In a comparative study involving serum samples from 64 fecal culture-positive cattle, the EVELISA identified 96.6% of the low-level fecal shedders and 100% of the midlevel and high-level shedders, whereas the Biocor ELISA detected 13.7% of the low-level shedders, 25% of the mid-level shedders, and 96.2% of the high-level shedders. Thus, the EVELISA was substantially superior to the Biocor ELISA, especially in detecting low-level and midlevel shedders. The EVELISA may form the basis for a highly sensitive and subspecies-specific test for the diagnosis of JD.
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