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.     

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.     

O2 Delivery and the Venous PO2-O2 Uptake Relationship in Pump-Perfused Canine Muscle

Under the conditions of both an increased red cell affinity for O(2) at a constant rate of O(2) delivery (arterial O(2) content x flow) and a decrease in the rate of O(2) delivery induced by hypoxic hypoxia at constant blood flow, we have obtained a linear relationship between the partial pressure of O(2) in the muscle venous effluent (P(v,)(O(2))) and O(2) uptake (.V(O(2))). The relationship is described by the equation .V(O(2)) = D(a) x P(v,)(O(2)) + .V(O(2)conv)) where D(a) is the apparent O(2) diffusion capacity and .V(O(2)conv)) is O(2) delivery-limited .V(O(2)), and D(a) x P(v,)(O(2)) represents the O(2) diffusion-limited .V(O(2)) .V(O(2)diff)). From these observations, we propose the hypothesis that .V(O(2)) consists of two additive values, .V(O(2)conv)) and .V(O(2)diff)). The mechanism underlying the reduction in .V(O(2)) that is induced by reducing O(2) delivery to markedly below the .V(O(2)conv)) value has only been investigated using a model based on the single compartment of diffusion-limited .V(O(2)), and has not been investigated in terms of this additive .V(O(2)) model. The single compartment analysis appears to overestimate the role of O(2) diffusion in limiting the reduction of .V(O(2)) that occurs in response to a decrease in O(2) diffusion capacity, as reflected by the .V(O(2))/P(v,)(O(2)) ratio. To gain better insight into the mechanism involved, we altered the rate of O(2) delivery by changing arterial P(O(2)) from normoxia (with inhalation of air) to hypoxia (by inhalation of 10-11 % O(2)) and blood flow (with high and low flow rates (n = 7 for both groups), and very low and ischaemic flow rates (n = 4 for both groups)) in pump-perfused dog gastrocnemius preparations during tetanic isometric contractions at 1 Hz. As rates of O(2) delivery were reduced from 23.2 to 10.9 ml min(-1) (100 g)(-1), significant decreases in P(v,)(O(2)) and .V(O(2)) were observed (P < 0.05). From the data of P(v,)(O(2)) and .V(O(2)) values within this range of O(2) delivery rates, we obtained the regression equation .V(O(2)) = 0.22 x P(v,)(O(2)) + 8.14 (r = 0.58). From the equation, the intercept of the .V(O(2))-axis was significantly different from zero (P < 0.05), in accordance with the observation that the .V(O(2)) /P(v,)(O(2)) ratio (ml min(-1) (100 g)(-1) Torr(-1)) increased from 0.54 to 1.35 (P < 0.05). However, at extremely low rates of O(2) delivery (5.6 and 7.3 ml min(-1) (100 g)(-1) the .V(O(2))/P(v,)(O(2)) ratio was 1.51 and 2.80 (P < 0.05), respectively. This indicates a break in the linear .V(O(2))-P(v,)(O(2)) relationship as the rate of O(2) delivery was reduced to below the .V(O(2)conv)) value of the .V(O(2))-axis intercept. These results suggest that the reduction in .V(O(2)) caused by extreme reductions in the rate of O(2) delivery is not attributable to a reduction in O(2) diffusion capacity, as expected from the .V(O(2))/P(v,)(O(2)) ratio, but to a reduction in the O(2) delivery-limited .V(O(2)) component, as evaluated by the .V(O(2))-axis intercept of the linear .V(O(2))-P(v,)(O(2)) relationship.     

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.     

Leu 7+(HNK-l+) Cells

In the present study, combined methods (indirect immunofluorescence with monoclonal antibodies, Percoll density fractionation, FACS analysis, and the cytotoxicity test) were used for further characterization of peripheral blood Leu 7+ cells (human NK and K cells). The Leu 7+ cell content was found to be relatively higher in the low-density cell fraction in which cells of large granular lymphocyte morphology predominated. However, Leu 7+ cells were also present in intermediate and high-density fractions. Low-density Leu 7+ cells were characterized by both Leu 2 (T suppressor/cytotoxic) and OKM1 (myelomonocytic) markers, whereas among high-density Leu 7+ cells the Leu 2 phenotype strictly predominated. Depletion of OKT3+ cells from the non-adherent cell population caused a decrease of cells with T helper and T suppressor phenotypes but did not have this effect on Leu 7+ and OKM1+ cells. After depletion of Leu 7+ cells from the OKT3- population the content of both T suppressor and OKM1+ cells decreased. Both the present results and previous reports enable us to conclude that two main Leu 7+ cell subpopulations are present in blood, namely Leu 7+Leu 2+/Leu 4+ and Leu 7+/OKM1+ cells. The presence of small and large Leu 7+ cells was also shown by FACS analysis.