Monoclonal antibodies for human and porcine histamine N-methyltransferase (HMT) facilitate protein expression and localization studies.

OBJECTIVE: The lack of suitable antibodies for the histamine inactivating enzyme histamine N-methyltransferase (HMT) has so far prevented the direct analysis of HMT proteins in man and other mammals. METHODS: A series of monoclonal antibodies was produced by immunizing mice with human and porcine HMT expressed in vitro. Antibodies were characterized by immunoblotting and immunohistochemical staining. RESULTS: Six different monoclonal antibodies specific for human HMT and four different monoclonal antibodies specific for porcine HMT were obtained that can detect HMT with up to tenfold greater sensitivity than the most sensitive enzymatic assays currently available. Using these antibodies allowed us to confirm the expression and cellular localization of HMT in various human and porcine tissues, where the presence of the enzyme had previously been deduced from activity measurement and HMT mRNA analysis. Immunohistochemical staining of human and porcine tissue sections clearly showed that HMT is a cytosolic protein, which is localized in specific cells of most mammalian tissues. CONCLUSIONS: The new monoclonal antibodies not only allow a comprehensive quantitative evaluation of the expression of HMT at the cellular level in man and other mammals but will also facilitate sensitive analyses of disease-associated alterations of this protein.

Association between two polymorphisms of histamine-metabolising enzymes and the severity of allergic rhinitis in a group of Mexican children

BACKGROUND: It has been suggested that polymorphisms of histamine metabolising enzymes can be a risk factor for developing histamine-involving diseases. The aim of the present study is to research the possible association between two functional single nucleotide polymorphisms (SNPs): C314T in the Histamine-N-Methyl Transferase gene and C2029G in the Diamine Oxidase gene, with the severity of allergic rhinitis and the number of allergic diseases, in a group of allergic Mexican children. METHODS: We studied 154 unrelated allergic children. SNPs were analysed by RT-PCR. The total serum IgE was measured by chemiluminescence and the serum histamine by ELISA. We used logistic regression analysis to determine OR. RESULTS: Patients carrying the mutant allele for any SNP had more risk to develop higher rhinitis severity or a bigger number of allergic diseases. Haplotype analysis revealed that this effect is synergistic. In patients carrying one or two mutant alleles, serum histamine levels were higher than those of patients carrying only wild alleles. Serum IgE levels were not associated with the presence of mutant alleles. CONCLUSION: The presence of these SNPs in patients with allergic rhinitis can lead to higher serum histamine, therefore to a higher risk of developing more severe symptoms or more associated allergic diseases, even if the serum IgE remains low

No disponible

Histamine N-methyltransferase 939A>G polymorphism affects mRNA stability in patients with acetylsalicylic acid-intolerant chronic urticaria.

BACKGROUND: Histamine plays an important role in allergic inflammation. Histamine levels are regulated by histamine N-methyltransferase (HNMT). OBJECTIVE: To investigate the functional variability of HNMT gene in relation to genetic polymorphisms in patients with aspirin intolerant chronic urticaria (AICU). METHODS: Two single-nucleotide polymorphisms of the HNMT gene (314C>T, 939A>G) were genotyped in chronic urticaria patients. The functional variability of 3'-untranslated region polymorphism (3'-UTR) was assessed using the pEGFP-HNMT 3'-UTR reporter construct to examine mRNA stability and fluorescence-tagged protein expression. The HNMT enzymatic activities related to the 939A>G polymorphism were examined both in the human mast cells (HMC-1) transfected with the pHNMT CDS-3'-UTR construct and in the patients' red blood cells (RBCs). Histamine release from the basophils of AICU patients was examined. RESULTS: The 939A>G polymorphism was significantly associated with the AICU phenotype, while no association was found with the 314C>T polymorphism. An in vitro functional study using HMC-1 cells demonstrated that the 939A allele gave lower levels of HNMT mRNA stability, HNMT protein expression, and HNMT enzymatic activity and higher histamine release than the 939G allele. The in vivo functional study demonstrated that the AICU patients with the 939A allele had lower HNMT activity in RBC lysates and higher histamine release from their basophils. CONCLUSION: The HNMT 939A>G polymorphism lowers HNMT enzymatic activity by decreasing HNMT mRNA stability, which leads to an increase in the histamine level and contributes to the development of AICU.

Tissue distribution of histamine N-methyltransferase-like immunoreactivity in rodents.

The rat kidney histamine N-methyltransferase was purified to homogeneity from Escherichia coli transfected with its recombinant cDNA. An antiserum to the enzyme was raised in rabbit by immunization with the purified protein. Western blot analysis of rat tissues with the antiserum revealed a band with identical mobility to that of purified enzyme in the extracts of kidney, jejunum, and brain, where the enzyme activity was detected. The antiserum cross-reacted with a 32K protein in mouse liver, brain, stomach, kidney and lung, and a 33K protein in guinea pig brain, stomach jejunum, spleen, lung, and kidney. The intensity of the staining in western blotting correlated well with the enzyme activity in all the tissues in these three species, suggesting that our antiserum is useful for quantifying histamine N-methyltransferase protein in rodent tissues.

A modified radioimmunoassay for the detection of histamine release in vivo and in vitro in man.

Histamine (Hi) release in vitro or in vivo in man was measured by variants of a radioimmunoassay (RIA) procedure. Hi released from isolated basophils was converted enzymically to N-tau-methylhistamine (NMH) which was then measured by a very sensitive RIA. This modified RIA was compared with the standard spectrofluorometric assay and was found to have additional advantages in certain applications. RIA of NMH in plasma was found to be of value in acute medical conditions of obscure aetiology.

Immunohistochemical demonstration of histamine N-methyltransferase-like structures in rat kidney.

Histamine N-methyltransferase (S-adenosylmethionine: histamine N-methyltransferase, E.C. 2.1.1.8) was purified to homogeneity from rat kidney, and antibody was raised against it in guinea pigs. The antibody immunoprecipitated histamine N-methyltransferase. Immunofluorescent histochemical studies with anti-histamine N-methyltransferase antibody as the first antibody and goat anti-guinea pig IgG conjugated with fluorescein isothiocyanate as the second, showed the presence of immunoreactive structures in the proximal tubules of rat kidney. The brain showed no immunoreaction with the antibody.

Rat histamine N-methyltransferase. Quantification, tissue distribution, purification, and immunologic properties.

Histamine N-methyltransferase is a major enzyme for inactivating histamine in mammalian tissues. Development of a sensitive and specific assay for histamine N-methyltransferase has permitted quantification of this activity in 25 rat tissues. Additionally, renal histamine N-methyltransferase was purified to electrophoretic homogeneity. The pivotal step in the purification scheme was an affinity column procedure which involved the specific elution of histamine N-methyltransferase from DEAE-Sephacel by 1 mM histamine. The purified enzyme has a molecular weight of 33,400 and a narrow pH dependency with an optimum at 8.0-8.25. Specific antibody produced to renal histamine N-methyltransferase also immunoprecipitated the brain enzyme completely, therefore indicating neuronal and non-neuronal histamine N-methyltransferase share common antigenic determinants.