A comparison of the biochemical properties of the human diaphorase (DIA3) isozymes determined by the common alleles DIA13, DIA23 and DIA33

(1) Various buffer systems for the starch gel electrophoresis of human diaphorase isozymes have been explored. Electrophoresis in a Tris/Borate system at pH 8.6 which includes 70 micron NADH in the gel and cathodal electrode buffers, provides good resolution of the six DIA3 phenotypes previously resolved by isoelectric focusing. (2) The variant genes DIA13, DIA23 and DIA33 occur with frequencies of about 0.76, 0.23 and 0.01 respectively in the English population. (3) The isozymes determined by the least common gene, DIA33, are markedly different from the isozymes determined by DIA13 and DIA23 in their relatively low heat stability, high affinity for Blue Sepharose and slow anodal electrophoretic mobility in buffer systems containing borate. The DIA3 1 and DIA3 2 isozymes are similar to one another in these characteristics.     

Assignment of a human diaphorase (DIA4) to chromosome 16

A human FAD-dependent diaphorase, DIA₄, has been studied in 29 independent human-rodent hybrids and in 17 subclones. The results suggest that the locus DIA, is on chromosome 16.     

Linkage relationships between the three phosphoglucomutase loci PGM1, PGM2 and PGM3

1. Phosphoglucomutase phenotypes have been studied in several generations of the family of an individual heterozygous at each of the three loci, PGM₁, PGM₂, and PGM₃. 2. PGM₁ and PGM₂ phenotypes were determined using red cells. Fibroblasts grown in tissue culture were used for PGM₃ phenotyping. 3. The family results support the genetical hypothesis based on the analysis of dizygotic twin pairs that the PGM₃ isozyme patterns found in the placenta are determined by two alleles, PGM¹₃ and PGM²₃. 4. Locus PGM₃ is not closely linked to locus PGM₂ 5. The data also support the previous findings that locus PGM₁ is not closely linked to PGM₂ or PGM₃.     

The relative activities attributable to the three phosphoglucomutase loci (PGM1, PGM,2 PGM3) in human tissues

1. The isozymes attributable to the three phosphoglucomutase loci, PGM_1;PGM₂ and PGM₃, have been separated by agarose-acrylamide gel electrophoresis and their relative activities have been measured in a range of human tissues. 2. In most tissues except red cells and fibroblasts, 85–95 % of the total PGM activity is determined by the PGM₁ locus, 2–15 % is contributed by the PGM₂ locus and 1–2 % is determined by the third locus PGM₃. 3. In fibroblasts the PGM₃ isozymes are relatively much more active and account for nearly 7 % of the total PGM activity. 4. In red cells approximately equal amounts of the PGM₁ and PGM₂ isozymes occur but no PGM₃ isozymes are found. 5. The atypical PGM isozyme pattern observed in red cells is probably a reflexion of in vivo stability differences between the three forms of PGM. In other tissues the PGM isozyme patterns are probably consequent upon differences in rates of synthesis or differences in the specific activities of the gene products.     

Human alcohol dehydrogenase ADH1, ADH2 and ADH3 loci in a mixed population of Bahia, Brazil

1. The three structural gene loci of human alcohol dehydrogenase have been studied in liver, jejunum and lung from 300 newborns in a triracially mixed population of Bahia, Brazil. 2. The frequency of the ADH23 allele was 0-1392, suggesting that the ADH23 allele is less frequent in Negroes. 3. A new ADH2 variant was identified. The electrophoretic pattern was interpreted as due to a new allele which is provisionally called ADH2Bahia. 4. By electrophoretic classification the 'atypical' variant was found in 2-8% of the sample. A question is raised regarding the ancestral origin of the 'atypical' variant in the population. Because this variant is common in Japanese it may have reached the present day population of Bahia through their American Indian ancestors. 5. Subjective estimation of the proportions of beta chains by giving scores to the liver isozymes alphaalpha, alphabeta and betabeta showed a clear relationship between the fetal weight and the beta chain activity. 6. The proportion of beta chains in the liver is significantly less when there is no enzyme activity in the lung, indicating some synchronous 'turning on' mechanism for alcohol dehydrogenase synthesis in both tissues.     

Assignment of the DIA1 locus to chromosome 22

Human/rodent hybrid cell cultures were examined for the presence of DIA1 and other marker enzymes. Many of these hybrids were also analysed for human chromosomes. Complete concordance was found only with chromosome 22.     

Evidence for the assignment of the loci AK1, AK3 and ACONs to chromosome 9 in man

The segregation of human enzymes and chromosomes has been studied in more than 30 independent primary human-rodent somatic cell hybrids and a series of 64 subclones. The results strongly suggest that the locus determining AK1, 'red cell' adenylate kinase, is on chromosome 9 in man, and hence that the locus for the ABO blood groups and that for the Nail-patella syndrome may also be assigned to this chromosome. Evidence is presented indicating that another adenylate kinase, nucleoside triphosphate adenylate kinase, and also the soluble form of aconitase, are probably syntenic with AK1, and that the mitochondrial form of aconitase is probably not syntenic with these loci.     

Regulation of histamine release from human basophil leucocytes: role of H1, H2 and H3 receptors

A novel class of histamine receptors (H3), controlling histamine synthesis and release, was described in rat and human brain and peripheral nerve endings. The present study was undertaken to evaluate whether H3 receptors contribute to the regulation of histamine release from human basophils. Basophil leucocytes were incubated with a H3 antagonist (thioperamide; concentrations ranging from 1 nM to 10 microM) or with a H3 ((R)alpha methyl-histamine; concentrations ranging from 1 to 100 mM), and subsequently were stimulated with optimal doses of anti-IgE and formyl-methionyl-leucyl-phenyl-alanine (f-met peptide). No significant modifications of histamine release were observed after incubation either with the H3 agonist or with the H3 antagonist. By contrast, a H2 antagonist (cimetidine; concentrations ranging from 1 to 100 microM) exerted a dose-dependent enhancing effect on anti-IgE- and, to a lesser extent, on f-met peptide-induced histamine release. A H1 antihistamine (chlorpheniramine; concentrations ranging from 100 nM to 1 microM), at the highest concentration employed, displayed an inhibitory activity on IgE-dependent and IgE-independent histamine release. Exogenous histamine was shown to exert a dose-dependent inhibitory effect on two-staged anti-IgE-induced histamine release. Taken as a whole, these results suggest that H3 receptors are not involved in the regulation of histamine release from human basophils; by contrast, H2 receptors participate in controlling histamine release from human basophils, as previously demonstrated by other authors.     

Identification of human phosphoglucomutase 3 (PGM3) as N-acetylglucosamine-phosphate mutase (AGM1)

We performed phenotyping of human phosphoglucomutase 3 (PGM₃) and screening for mutations in the human N‐acetylglucosamine‐phosphate mutase gene (AGM₁) to identify PGM₃ as AGM₁. By sequencing the coding region of AGM₁, two alleles containing a G or A base at nucleotide 1396, that can respectively encode aspartic acid or asparagine at codon 466, were identified. Cell extracts of COS7 cells after transfection with the pcDNA 3·1(+) plasmid containing an AGM₁ allele with 1396G or 1396A showed similar electrophoretic patterns to the PGM₃ 1 or PGM₃ 2 protein, respectively, with the isozyme detection method used for PGM₃ phenotyping. The genotypes determined by the two alleles of AGM₁ coincided exactly with the PGM₃ phenotypes in 20 individuals. We also investigated the allele frequency of the AGM₁ nucleotide polymorphism in a Japanese population by DNA sequencing and found that the frequencies of alleles 1396G and 1396A were similar to previously reported PGM₃*1 and PGM₃*2 frequencies. Overall, the facts that the AGM₁ gene product shows PGM activity, AGM₁ is polymorphic, the electrophoretic mobility is similar between AGM₁ allele‐specific products and PGM₃ 1 and 2 proteins, PGM₃ phenotypes and AGM₁ genotypes completely coincide in 20 individuals, and AGM₁ allele frequencies are similar to those of PGM₃*1 and PGM₃*2 in Japanese populations, suggest that PGM₃ is identical to AGM₁.     

Aquaporin-1 and HCO3−-Cl− transporter-mediated transport of CO2 across the human erythrocyte membrane

Recent studies have suggested that aquaporin-1 (AQP1) as well as the HCO₃⁻-Cl⁻ transporter may be involved in CO₂ transport across biological membranes, but the physiological importance of this route of gas transport remained unknown. We studied CO₂ transport in human red blood cell ghosts at physiological temperatures (37 °C). Replacement of inert with CO₂-containing gas above a stirred cell suspension caused an outside-to-inside directed CO₂ gradient and generated a rapid biphasic intracellular acidification. The gradient of the acidifying gas was kept small to favour high affinity entry of CO₂ passing the membrane. All rates of acidification except that of the approach to physicochemical equilibrium of the uncatalysed reaction were restricted to the intracellular environment. Inhibition of carbonic anhydrase (CA) demonstrated that CO₂-induced acidification required the catalytic activity of CA. Blockade of the function of either AQP1 (by HgCl₂ at 65 μM) or the HCO₃⁻-Cl⁻ transporter (by DIDS at 15 μM) completely prevented fast acidification. These data indicate that, at low chemical gradients for CO₂, nearly the entire CO₂ transport across the red cell membrane is mediated by AQP1 and the HCO₃⁻-Cl⁻ transporter. Therefore, these proteins may function as high affinity sites for CO₂ transport across the erythrocyte membrane.     

Ganglioside GM2 N-acetyl-ß-D-gafactosaminidase and asialo GM2 (GA2) N-acetyl-ß-D-galactosaminidase; studies in human skin fibroblasts

Ganglioside G_M2 and its asialo-derivative, G_A2 were radiolabeled in their N-acetyl-D-galactosaminyl moieties by oxidation with galactose oxidase and reduction with tritiated sodium borohydride. Specific activities of 6 × 10⁴ dpm/nmol (G_M2) and 1.8 × 10⁶ dpm/nmol (G_A2) were achieved. About 98% of the label was in N-acetyl-D-galactosamine. Using these substrates, an assay was developed for G_M2-N-acetyl-β-D-galactosaminidase (E.C.3.2.1.30) and G_A2-N-acetyl-β-D-galactosaminidase (E.C.3.2.1.30) activities in human cultured skin fibroblasts. The products of the G_M2 cleaving reaction were identified as N-acetylgalactosamine and ganglioside G_M3- Both G_M2 and G_A2 cleaving activities were stimulated about 5-fold by purified sodium taurocholate, and this stimulation was inhibited by neutral detergents, lipids and albumin at low concentrations. Addition of various salts, reducing agents and a protein activator factor from human liver of Li et al. (1973) did not stimulate G_M2-N-acetyl-β-D-galactosaminidase activity beyond that found with sodium taurocholate. Under optimal conditions, control fibroblast supernates cleaved ganglioside G_M2 at a rate of 3.7 nmol/mg protein/h compared to 1100 for G_A2-N-acetyl-β-D-galactosaminidase and 4700 for 4-methylumbelliferyl-N-acetyl-β-D-glucosaminidase. Supernates from two patients with Tay-Sachs disease had markedly reduced activity levels for G_M2-N-acetyl-β-D-galactosaminidase but not for the other two substrates. Supernates from two patients with Sandhoff's disease had reduced activities for all three substrates. A supernate from one patient with juvenile G_M2 gangliosidosis cleaved G_M2 at a somewhat faster rate than those from Tay-Sachs or Sandhoff's patients. Two healthy adult women with markedly reduced hexosaminidase A activities using 4MU-N-acetyl-β-D-glucosaminide as substrate had approximately half-normal activities using G_M2 as substrate. A patient with the Tay-Sachs phenotype but with a partial deficiency of hexosaminidase A using the 4-MU substrate had a profound deficiency using G_M2 as substrate. In such unusual hexosaminidase mutants, assays using G_M2 as substrate are better indicators of phenotype than those using synthetic substrates.     

Reactivity to sodium tetrachloropalladate (Na2[PdCl4]) compared to PdCl2 and NiCl2 in lymphocyte proliferation tests

Background: For patch testing, replacement of the commonly used palladium dichloride (PdCl₂) by sodium tetrachloropalladate (Na₂[PdCl₄]) was recently demonstrated to improve test accuracy and show a significant correlation with nickel (Ni), supporting the concept of cross‐reactivity between Pd and Ni. A promising alternative to metal allergy patch testing is the in vitro lymphocyte proliferation test (LTT). Objectives: The aim of this study was to test whether Na₂[PdCl₄] is also more sensitive for diagnosing Pd allergy with a standardized LTT. Patients/methods: After determining optimal nontoxic and nonmitogenic concentrations for Na₂[PdCl₄], blood samples from 105 patients with clinical suspicion of metal allergy were tested with an LTT called memory lymphocyte immuno stimulation assay for Na₂[PdCl₄], PdCl₂ and NiCl₂. Reaction profiles were analysed for concordant positive reactions. Results: Using the conventional cut‐off of stimulation index ≥ 3, 74.3% showed a positive reaction to NiCl₂, 15.2% to PdCl₂ and 28.6% to Na₂[PdCl₄]. All positive results to PdCl₂ were covered by Na₂[PdCl₄]. From the 30 positive reactions to Na₂[PdCl₄], 26 (87%) were concordant for NiCl₂ reactivity. Conclusion: In LTT, the use of Na₂[PdCl₄] results in more positive reactions in Pd allergy testing which are in concordance with positive reactions to PdCl₂ and NiCl₂.