A human dispermic chimaera first suspected from analyses of the blood group gene-specified glycosyltransferases

The red cells of a normal male blood donor, K.S., were first grouped as B but he was found to lack anti-A in his serum. Closer investigation revealed that his red cells had very weak A activity, demonstrable only by absorption and elution of anti-A. He is a non-secretor of ABH and a secretor of Lea. Blood group A-, B and H-gene specified glycosyltransferases were detected in his serum. In contrast to the finding of a B antigen of normal strength on his red cells, the B transferase in his serum was only about 30% of the normal level and, despite the very weak A activity of K.S's red cells, the A transferase level was about 50% of that found in the serum of group A individuals with normal strength of A antigen. Moreover, the A transferase on the basis of its pH optimum, Km values for donor and acceptor substrates, activation by divalent cations, isoelectric focusing profile and capacity to convert O to A-active cells, was characterized as the product of an A1 gene. A family study showed that K.S's wife is group A2 and that they have two sons, one group A2 and the other group B. The group B son is assumed to have inherited a B gene from the propositus but the level of B transferase in the son's serum is three times as high as that in his father's serum. The wife of the propositus and his group A2 son have normal A2 transferases in keeping with their A2 red cell status. The A2 son therefore appears to have inherited an A2 gene from his mother but neither the A1 nor the B gene shown to be carried by his father. The distribution of transferase activities in K.S's red cells differs from that in his serum. A level of B transferase within the normal range was found in his red cell membranes but a very low level of A transferase was detected. The discrepancies between the serum transferases and ABO red cell group, together with the pattern of inheritance within the family, led to a suspicion of chimaerism. This was confirmed by the finding of fibroblasts with the female 46XX karyotype in cultures of the propositus' skin. These results suggest that K.S. is a dispermic chimaera with two different cell lines of the genotypes BO and A1O or A1A1. The group A2 son is assumed to have inherited an O gene from his father. It seems probable that K.S.'s bone marrow and reproductive organs are comprised predominantly of the XY cell line which carried the blood group BO genotype whereas his skin and other tissues which contribute the A1 transferase to his plasma, are partly made up of the XX cell line which carries the blood group A1O or A1A1 genotype.     

DETECTION OF A1A2 AND A2AAm1 HETEROZYGOTES AMONG HUMAN A BLOOD GROUP PHENOTYPES

From the variations of α-N-acetylgalactosaminyltransferases activities with the pH, evidence was obtained for the recognition of A₁A₂ heterozygotes in normal A blood group sera. Besides, unusual transferase properties associated with two A₂ sera from individuals out of A^A_m1 siblings, lead to the identification of the very infrequent A₂A^A_m1 genotypes. These results strongly support the simultaneous coexistence of both A₁ and A₂ transferases in heterozygotes' sera, and bring some new information on the genetical background of the A_m phenotype. The meaning of transferase properties directly determined on whole sera is briefly discussed.     

THE cis AB PHENOTYPE IN THREE GENERATIONS OF ONE FAMILY: SEROLOGICAL,ENZYMATIC AND CYTOGENETIC STUDIES

The blood from three individuals belonging to consecutive generations of one family were characterized as cis AB on the basis of (a) the serological behaviour of the B antigen on their red cells, (b) the presence of weak anti-B in their sera and (c) family studies, which unequivocally demonstrated the genotype AB: O for two of the individuals with atypical AB groups. The serological behaviour of the A antigen on the red cells was intermediated between that of normal A₁ and A₂ cell. The B antigen gave weak and variable reactions with naturally occurring antibodies. Treatment of the cis AB red cells with an α-galactosyltransferase in the serum from an A₁B individual rendered them agglutinable by the normal range of anti-B sera, demonstrating that the red cells do not lack precursors for the formation of normal B-active structures. The cis AB sera all contained relatively strong α-N-acetylgalactosaminyl-transferases which had pH optima of 6.0, characteristic of A₁ gene-specified enzymes. The affinity of the enzymes for UDP-galactose, as measured by the apparent Ki, appeared to be greater than that of the A transferases in normal A₁ and A₁B sera. The B-gene transferases in the cis AB sera were very weak and were more readily inhibited by UDP-N-acetylgalactosamine than were the α-galactosyltransferases in normal B and AB sera. Both the A- and B-transferase activities in the cis AB sera are, therefore, in some ways different from the corresponding enzyme activities in the sera of normal A, B or AB donors. Cytogenetic study of blood lymphocytes from all three individuals showed normal karyotypes. Specifically, the terminal bands of the long arms of the No. 9 chromosomes, to which the ABO:Np-1: AK-1 linkage group has been assigned, displayed G bands similar to those in cells from other (normal) individuals examined simultaneously.     

Reaction of 7S and 19S Components of Immune Rabbit Antisera with Human Group A and AB Red Cells

7S and 19S components of rabbit anti-A antibody, labelled with ¹³¹I have been separated by chromatography on DEAE-cellulose. The reactions of these components with human group A₁, A₂ and AB red cells have been investigated. At saturation point A₁ cells combine with 0.22 μg. of 7S antibody per million cells, this corresponds to a minimum of 8.3×10⁵ A-antigen sites per cell. A₂ cells can take up only one quarter as much antibody, and combine much less avidly with it than do A₁ cells. Cells can take up only about one-fifth as many 19S as 7S antibody molecules. 19S antibody is more avid and, on a molecular basis, is 750 times more efficient at agglutinating red cells than 7S antibody. The significance of these findings is discussed with regard to the differences between A₁ and A₂ antigens, and the valency and mode of attachment to the red cells of the antibodies.     

α-N-ACETYL-d-GALACTOSAMIYL- AND α-d-GALACTOSYLTRANSFERASE ACTIVITIES IN SERA OF CIS AB BLOOD GROUP INDIVIDUALS

Thirteen cis AB persons from five families were examined for serum glycosyltransferase activities associated with the biosynthesis of A and B blood group characters. Their transferases were generally homogeneous within one family, except for A₂/cis AB genotypes, whose A enzyme level was similar to the A₂ normal sera, but they varied from one family to another. These activities differed quantitatively and qualitatively from A, B and AB normal sera. Studies of A transferase showed variations in the pH-dependent curve, the effect of cofactors and the capacity of conversion of O red cells into A-active cells. Moreover, A and B transferases behaved differently with respect to their relative levels than did AB heterozygous normal sera. The results were discussed and it was suggested that a mutation of a single enzyme transferring both galactose and N-acetyl-galactosamine could explain these properties.     

Analysis of Ligand-Receptor Interactions from the Molecular Level to the Whole-Body Level

A system for the quantitative analysis of ligand-receptor interactions is presented, based on models of different levels of complexity. For two pools of receptors, binding of a radioactive ligand is described by b = [(B _m1·A ⁿ¹)/(K _d1 ⁿ¹ + A ⁿ¹)] + [(B _m2·A ⁿ²)/(K _d2 ⁿ² + A ⁿ²)], (1)where b is the number of bound receptors at a ligand concentration [A], B _m1 and B _m2 are the receptor concentrations, K _d1 and K _d2 are dissociation constants for the ligand-receptor complex, and n1 and n2 are Hill coefficients. The magnitude of the physiological response for a system consisting of two discrete pools of receptors with different affinities is given by p = [(P _m1·A ⁿ¹)/(EC50₁ ⁿ¹ + A ⁿ¹)] + [(P _m2·Aⁿ²)/(EC50₂ ⁿ² + A ⁿ²)], (2)where p is the magnitude of the response to an agonist (or antagonist) at concentration [A], P _m1 and P _m2 are the maximal magnitudes of the responses for the individual pools of receptors, EC50₁ and EC50₂ are the agonist concentrations giving responses of magnitudes P _m1/2 and P _m2/2, and n1 and n2 are Hill coefficients. The parameters of these equations show: the number of pools of receptors with different affinities for the ligand (K _d or EC50), the number of active receptors (B _max) or the magnitudes of the maximal response (P _max), and the numbers of ligand molecules binding with the receptor (n, the Hill coefficient). E is the efficiency (E = B _max/2K _d, or E = P _max/2EC50) and gives the overall characteristics of the activity of the effector system. This method of analysis can be applied to any biological reactions whose results can be presented quantitatively.     

A2 adenosine receptors in Mongolian gerbil middle ear epithelium and their regulation of Cl− secretion

The present study investigates the effects of adenosine and its analogues on Cl^? secretion in primary cultures of gerbil middle ear epithelium. Short-circuit current (I_sc), an index of transepithelial active transport, was measured on the same cells cultured on porous filters. Baseline I_sc and transepithelial resistance were 27.0 ± 0.7 μA cm^?2 and 275 ± 7 Ω cm², respectively (n = 178). Extracellular adenosine and its analogues elicited a sustained increase in I_sc when added to apical or basolateral surfaces. Both the A_2A selective agonist 2-p-(2-carboxyethyl)phenethylamino-5′-N-ethylcarboxamido adenosine and the A_2A/A_2B nonselective agonist 5'-(N-ethyl-carboxamido)adenosine (NECA) increased I_sc, but NECA was more effective than CGS21680. A₁ selective antagonist 8-cyclopentyl-1,3-dipropylxanthine did not reduce NECA-induced I_sc. These results suggest the presence of both A_2A and A_2B receptors. NECA did not stimulate a rise in the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in single middle ear epithelial cells cultured on glass coverslips. Dibutyryl cAMP (dbcAMP) induced an initial transient increase in I_sc followed by the sustained plateau. Addition of dbcAMP also caused a transient increase in [Ca²⁺]_i. The protein kinase A inhibitor, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide, greatly reduced the increase in the I_sc responses to NECA. 1,2-Bis-(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid-acetoxymethyl ester influenced neither the NECA-induced increase in I_sc nor the dbcAMP-induced sustained phase of I_sc, but greatly inhibited the dbcAMP-induced transient increase in I_sc. Glibenclamide, a cystic fibrosis transmembrane conductance regulator (CFTR) channel inhibitor, reduced the NECA-induced I_sc. These results indicate that extracellular adenosine and its analogues activate the cAMP-protein kinase A system, but not intracellular Ca²⁺-dependent mechanisms, leading to Cl^? secretion, possibly through the CFTR Cl^? channels in the cultured gerbil middle ear epithelium.     

Adenosine A2B Receptors on Cardiac Stem Cell Antigen (Sca)-1–Positive Stromal Cells Play a Protective Role in Myocardial Infarction

Transplantation of mesenchymal stem-like cells to the heart is known to improve cardiac recovery in animal models of myocardial infarction (MI). Because stimulation of A_2B adenosine receptors on mouse cardiac stem cell antigen (Sca)-1⁺CD31⁻ mesenchymal stem-like cells significantly up-regulates their secretion of pro-angiogenic factors, we hypothesized that ablation of the A_2B receptor signaling in these cells would reduce their ability to improve vascularization of the infarct area seen after transplantation. Wild-type (WT) C57BL/6 mice underwent permanent left coronary artery ligation and received intramyocardial injections of Sca-1⁺CD31⁻ cells generated from WT or A_2B receptor knockout (A_2BKO) mice or the same volume of cell-free saline. Only 12% to 16% of injected cells remained in the ventricles 1 week later; there was no significant difference between WT and A_2BKO cell survival. Transplantation of WT, but not A_2BKO, cells significantly reduced both post-MI decline in cardiac function and adverse remodeling compared with that seen in control hearts. Morphological analysis conducted 4 weeks after MI revealed significantly increased vascularization of the infarct areas and reduced myocardial scarring in animals treated with WT, but not with A_2BKO, cells compared with control. Thus, our study demonstrated that the A_2B receptor signaling linked to up-regulation of pro-angiogenic factors in cardiac Sca-1⁺CD31⁻ stromal cells is essential for overall improvement of cardiac recovery seen after their transplantation to the injured heart.Mesenchymal stem-like cells of various tissue origins have been proposed to be used in cell-based transplantation therapy to enhance tissue repair and functional recovery after myocardial infarction (MI). Among them, cardiac mesenchymal stem-like cells have been consistently shown to possess superior paracrine potency and myocardial protection efficacy compared with stem/progenitor cells originated from other tissues.^1,2 In the mouse heart, these cells are represented by a population of stromal cells characterized by the expression of stem cell antigen (Sca)-1 on their surface and the absence of the endothelial cell surface marker, CD31.^3–9 Several groups independently reported that the delivery of cardiac Sca-1⁺CD31⁻ cell populations to the heart resulted in improved revascularization of injured tissue and attenuated decline of cardiac function in animal models of MI.^6,9–11 Although the precise mechanism of these protective effects remains unknown, the early assumption that these cells can replace damaged cardiomyocytes has recently given way to the realization that they also, and perhaps mainly, exert a beneficial effect via the release of paracrine factors.^3,12–17 As such, the beneficial properties of transplanted cells are likely regulated by local factors present in the ischemic tissue, including high levels of extracellular adenosine.Adenosine is an endogenously produced signaling molecule that binds to the family of extracellular G-protein–coupled seven transmembrane receptors, which include A₁, A_2A, A_2B, and A₃ subtypes. Adenosine concentrations in the myocardial interstitium remain low in normal conditions. Myocardial ischemia is known to considerably increase extracellular adenosine levels.^18,19 MI results in even larger levels of extracellular adenosine because of its massive release from damaged cells and catabolism of coreleased adenine nucleotides. Subsequent inflammation in the injured myocardium may further increase extracellular adenosine levels as the result of cell stress and tissue hypoxia.²⁰ Therefore, extracellular adenosine becomes an important part of the tissue microenvironment generated by myocardial ischemia and infarction.Recently, we found that stimulation of A_2B adenosine receptors on mouse cardiac Sca-1⁺CD31⁻ stromal cells significantly increased secretion of pro-angiogenic factors.²¹ In this study, we tested the hypothesis that the A_2B receptor signaling in these cells is important for their therapeutic effects seen after transplantation to the heart after MI.     

Extrachromosomal genetics in the yeast Kluyveromyces lactis. Isolation and characterization of antimycin-resistant mutants

Summary Antimycin-resistant (A^R) mutants of the yeast Kluyveromyces lactis, obtained either spontaneously or after manganese treatment, were isolated and genetically characterized. Most of the mutants obtained after manganese mutagenesis and two spontaneous mutants, tolerated high antimycin concentrations (more than 10 /gmg/ml) and were extrachromosomal. One mutant which grew only in low antimycin (1 /gmg/ml) showed a Mendelian type of inheritance. The extrachromosomal mutants could be assigned to at least two genetic loci (A _I ^R and A _II ^R ). Mutants representative of these two groups showed increased resistance to the antibiotic when the respiration of whole cells or mitochondria was studied. Extrachromosomal mutants of Saccharomyces cerevisiae resistant to antimycin were also induced with manganese, isolated and characterized. Comparative studies of the antimycin-resistant mutants of K. lactis and S. cerevisiae permitted the following observations: a) K. lactis is more resistant to antimycin, funiculosin, mucidin and diuron than S. cerevisiae, as are the A^R mutants; b) K. lactis shows correlated sensitivity to funiculosin differing in this aspect from S. cerevisiae; c) the antimycin-resistant mutants of K. lactis belonging to group 11 (A _II ^R ) were also resistant to diuron, tolerating concentrations of more than 200 /gmg/ml; d) all extrachromosomal antimycin-resistant-mutants of S. cerevisiae and some of the A^R mutants of K. lactis were more sensitive to mucidin than the wild type.Abbreviations diuron or DCMU 3-(3,4-Dichlorophenyl)-1,1dimethylurea - HQNO 2-n Heptyl-4-hydroxyquinoline N-oxide     

Study of stereoregularity of poly(vinyl ketone)s by proton nuclear magnetic resonance

Two poly(vinyl ketone)s, poly(methyl vinyl ketone) (PMVK) and poly(phenyl vinyl ketone) (PPVK), are isotacticly synthesized. The ¹H-NMR spectra of these polymers and of the meso and racemic model dimers help to determine the percentage of isotactic dyads using the area of the peaks of the non equivalent protons (H_A and H_B) of the β-methylene group of the chain. The difference between the chemical shifts of the H_B and H_B protons is exceptionally large (approximately 0,8 ppm for both isotactic PMVK and isotactic PPVK). The coupling constants and the β-CH₂ and the α-CH groups' frequencies are determined by applying the ABX₂ system. A spontaneous epimerization of isotactic PPVK is found; this is always accompanied by a degradation and leads to the assumption of a simultaneous photochemical reaction of the Norrish II type.     

39K and 23Na relaxation times and MRI of rat head at 21.1 T

At ultrahigh magnetic field strengths (B₀ ≥ 7.0 T), potassium (³⁹K) MRI might evolve into an interesting tool for biomedical research. However, ³⁹K MRI is still challenging because of the low NMR sensitivity and short relaxation times. In this work, we demonstrated the feasibility of ³⁹K MRI at 21.1 T, determined in vivo relaxation times of the rat head at 21.1 T, and compared ³⁹K and sodium (²³Na) relaxation times of model solutions containing different agarose gel concentrations at 7.0 and 21.1 T. ³⁹K relaxation times were markedly shorter than those of ²³Na. Compared with the lower field strength, ³⁹K relaxation times were up to 1.9‐ (T₁), 1.4‐ (T_2S) and 1.9‐fold (T_2L) longer at 21.1 T. The increase in the ²³Na relaxation times was less pronounced (up to 1.2‐fold). Mono‐exponential fits of the ³⁹K longitudinal relaxation time at 21.1 T revealed T₁ = 14.2 ± 0.1 ms for the healthy rat head. The ³⁹K transverse relaxation times were 1.8 ± 0.2 ms and 14.3 ± 0.3 ms for the short (T_2S) and long (T_2L) components, respectively. ²³Na relaxation times were markedly longer (T₁ = 41.6 ± 0.4 ms; T_2S = 4.9 ± 0.2 ms; T_2L = 33.2 ± 0.2 ms). ³⁹K MRI of the healthy rat head could be performed with a nominal spatial resolution of 1 × 1 × 1 mm³ within an acquisition time of 75 min. The increase in the relaxation times with magnetic field strength is beneficial for ²³Na and ³⁹K MRI at ultrahigh magnetic field strength. Our results demonstrate that ³⁹K MRI at 21.1 T enables acceptable image quality for preclinical research. Copyright © 2016 John Wiley & Sons, Ltd.     

Loss of <Emphasis Type="Italic">p16</Emphasis><Superscript><Emphasis Type="Italic">INK4A</Emphasis></Superscript> expression is associated with allelic imbalance/loss of heterozygosity of chromosome 9p21 in microdissected synovial sarcomas

Deletions of the short arm of chromosome 9 have been observed in many tumours and cell lines. This chromosomal region is frequently targeted during malignant transformation because it contains at least two known tumour suppressor genes: p16 ^INK4 and p15 ^INK4B . p16 ^INK4A acts as a negative cell cycle regulator by inhibiting G₁ cyclin-dependent kinases that phosphorylate the retinoblastoma protein and therefore block the progression of the cell cycle from G₁ to S phase. The role of p16 ^INK4A in the development of synovial sarcoma has not been comprehensively investigated. Ten samples of synovial sarcomas were examined for allelic imbalance/loss of heterozygosity (AI/LOH) of the 9p region and p16 protein expression. DNA was isolated from microdissected sections of normal and tumour cells, amplified by polymerase chain reaction and analysed for AI/LOH by using six microsatellite markers that map to the 9p region. Immunohistochemistry for p16 expression was done. AI/LOH with at least one microsatellite marker on 9p21 was detected in six of ten samples. The most frequent allelic deletions were observed within the coding sequence of p16 ^INK4A . Loss of p16 immunoreactivity was detected in eight samples, six of which showed evidence of alterations at 9p21 region. These findings suggest a possible role of loss of p16 ^INK4A in the development of synovial sarcoma.     

Enteric glial adenosine 2B receptor signaling mediates persistent epithelial barrier dysfunction following acute DSS colitis

Intestinal epithelial barrier function is compromised in inflammatory bowel disease and barrier dysfunction contributes to disease progression. Extracellular nucleotides/nucleosides generated in gut inflammation may regulate barrier function through actions on diverse cell types. Enteric glia modulate extracellular purinergic signaling and exert pathophysiological effects on mucosal permeability. These glia may regulate inflammation with paracrine responses, theoretically mediated via adenosine 2B receptor (A_2BR) signaling. As the cell-specific roles of A_2BRs in models of colitis and barrier dysfunction are unclear, we studied glial A_2BRs in acute dextran sodium sulfate (DSS) colitis. We performed and validated conditional ablation of glial A_2BRs in Sox10^CreERT2+/?;Adora2b^f/f mice. Overt intestinal disease activity indices in DSS-colitis were comparable between Sox10^CreERT2+/–;Adora2b^f/f mice and littermate controls. However, ablating glial A_2BRs protected against barrier dysfunction following acute DSS-colitis. These benefits were associated with the normalization of tight junction protein expression and localization including claudin-1, claudin-8, and occludin. Glial A_2BR signaling increased levels of proinflammatory mediators in the colon and cell-intrinsic regulation of genes including Csf3, Cxcl1, Cxcl10, and Il6. Our studies show that glial A_2BR signaling exacerbates immune responses during DSS-colitis and that this adenosinergic cell-specific mechanism contributes to persistent gut epithelial barrier dysfunction.     

INHERITED ‘MOSAICISM’ WITHIN THE ABO BLOOD GROUP SYSTEM

A family with examples of the rare condition known as ‘inherited mosaicism affecting the ABO blood groups’ has been studied. In this family there were five examples of B_mos:O mosaicism. Blood group gene-specified transferase estimations were studied in this condition for the first time. In ‘affected' members, levels of B gene-specified transferease were low, and amounted to only 7-10% of the activity of a normal control. It is proposed that B_mos is correctly classified as a new form of weak B.     

肺炎衣原体对C57BL/6J小鼠腹膜巨噬细胞泡沫化的影响

目的:探讨肺炎衣原体在C57BL/6J小鼠腹膜巨噬细胞泡沫化过程中的作用。方法:将肺炎衣原体与C57BL/6J小鼠腹膜巨噬细胞培养72h后用油红O方法染色观察细胞的形态变化并测定细胞内胆固醇的含量。结果:肺炎衣原体能使C57BL/6J小鼠腹膜巨噬细胞胞浆内脂质颗粒增多,胆固醇酯占总胆固醇的比例增加。结论:肺炎衣原体能促进C57BL/6J小鼠腹膜巨噬细胞泡沫化。     

B-COMPLEX GENETIC CONTROL OF IMMUNE RESPONSE TO HSA, (T,G)-A–L,GT AND OTHER SUBSTANCES IN CHICKENS

Immune response to poly-(l -tyrosine-l -glutamic acid)-poly-d , l -alanine-poly-l -lysine ((T,G)-A–L), human serum albumin (HSA), and (l -glutamic acid⁵⁰, l -tyrosine⁵⁰)n (GT) was found to be linked to the B complex in an outbred line of Leghorns segregating for the B¹, B², and B¹⁹ alleles. Birds of the blood group genotypes B¹B¹, B²B², and B¹⁹B¹⁹ were low, intermediate, and high responders, respectively to either (T,G)-A–L or HSA. Response to GT, however, differed, with the B²B² genotype being the only responder. No real genotype differences in immune response to DNP-conjugates and sheep red blood cells (SRBC) could be detected.     

Early interactions of foot-and-mouth disease virus with cultured cells

The adsorption of foot-and-mouth disease virus (FMDV) types A₁₂119, O_1B, and C_3Res were studied in baby hamster kidney (BHK-21) cells by measuring the amount of radioactively labeled purified virus which remained attached to cells at various times after infection. At 4°, over half of the maximum adsorption of type A virus occurred within 15 min, and approximately 65% of the radioactivity bound by 45 min was removed by brief treatment with EDTA, indicating the assay measures primarily adsorption and not penetration. Upon shifting the temperature from 4 to 37°, about 60 to 70% of the bound virus eluted by 1 hr in an unmodified form. Only 140 S virus particles and 12 S subunits could be found associated with the infected cell 20 min after shifting the temperature from 4 to 37°. By 50 min, the number of 140 S particles decreased slightly. The number of viral receptors per BHK-21 cell was determined to be 1–2.5 × 10⁴ for virus types A₁₂ and O_1B. Competition experiments between FMDV types A₁₂, O_1B, and C_3Res indicated that they can utilize at least some common receptor sites on cells.     

Isolation of a lactic dehydrogenase-A-deficient CHO-K1 mutant by nylon cloth replica plating

A mutant Chinese hamster ovary cell deficient in lactate dehydrogenase A activity has been isolated using a nonselective technique. The method uses histochemical staining to examine colonies directly for enzyme activity and nylon cloth replica plating to recover particular clones. The mutant cell has an apparent K_m (pyruvate to lactate) that is nearly tenfold higher than the parental cell, while itsV _max has been reduced more than 80-fold. In mutant cell extracts with added porcine LDH-B enzyme, molecular dissociation and recombination of subunits produces two new active LDH tetramers (A₁B₃, A₂B₂). The electrophoretic mobility of at least one of the tetramers (A₁B₃) was different from those formed in the parental extracts. The evidence suggests the variant cell contains a mutation in the structural gene for LDH-A.     

Adenosine is not a direct GHSR agonist--artificial cross-talk between GHSR and adenosine receptor pathways

Aim: To assess if adenosine is a direct growth hormone secretagogue receptor (GHSR) agonist by investigating the mechanism behind adenosine induced calcium release in human embryonic kidney 293s (HEK) cells expressing GHSR. Methods: Calcium mobilization, cyclic adenosine monophosphate (cAMP) and IP₃ experiments were performed using HEK cells stably expressing GHSR and/or adenosine A_2B receptor (A_2BR). Results: Adenosine has been widely reported as a GHSR agonist. In our hands, adenosine and forskolin stimulated calcium release from IP₃ controlled stores in HEK–GHSR cells but not in non‐transfected HEK cells. This release was not accompanied by increased IP₃ levels. The calcium release was both cholera toxin and U73122 sensitive, indicating the involvement of both Gα_s/adenylyl cyclase and Gα_q/11/phospholipase C pathways. Importantly, the GHSR inverse agonist [D‐Arg¹ D‐Phe⁵ D‐Trp^7,9 Leu¹¹]‐Substance P (SP‐analogue) blocked the adenosine stimulated calcium release, demonstrating that GHSR is involved. Assessment of the GHSR‐dependent calcium release using adenosine receptor agonists and antagonists resulted in a rank order of potencies resembling the profile of A_2BR. A_2BR over‐expression in HEK–GHSR cells enhanced potency and efficacy of the adenosine induced calcium release without increasing IP₃ production. Moreover, A_2BR over‐expression in HEK cells potentiated NECA‐induced cAMP production. However, GHSR expression had no effect on intracellular cAMP production. Conclusion: In HEK–GHSR cells adenosine activates endogenously expressed A_2BR resulting in calcium mobilization. We hypothesize that the responsible mechanism is cAMP‐dependent sensitization of IP₃ receptors for the high basal level of IP₃ caused by GHSR constitutive activity. Altogether, our results demonstrate that adenosine is not a direct GHSR agonist.     

Studies on the quantitative variation of human red cell peptidase A activity

1. A new method for the assay of peptidase A activity in human red cells and leucocytes is described. 2. Wide variation in the level of red cell peptidase A activity between different individuals with the common electrophoretic phenotype Pep A1 has been demonstrated. 3. Family studies show highly significant parent-child and sib-sib correlations, but no significant parent-parent correlation. The findings indicate that the differences in level of activity between individuals are to a large extent genetically determined. 4. Peptidase A activities in leucocytes show much less variation than in red cells. Only a small correlation, which was not statistically significant was found between red cell and leucocyte activity. In particular the levels of leucocyte peptidase A activity in individuals with very weak red cell activities were found to be very similar to those seen in individuals with strong red cell activity. 5. Weak peptidase A activity in red cells was not found to be associated with weak activity of other red cell peptidases. 6. No evidence for the differential occurrence of inhibitors or activators of peptidase A in red cells of individuals with different levels of activity was obtained in various experiments designed to test this possibility. 7. The electrophoretic patterns seen in individuals heterozygous for certain rare alleles (Pep A⁵ and Pep A⁶) which determine electrophoretic variants of peptidase A suggest that two different alleles (provisionally called Pep A^1S and Pep A^1W) determining different forms of the enzyme with the usual electrophoretic mobility (Pep Al), but with different activities in red cells occur. Individuals presumed to be heterozygous for Pep A⁵ (or Pep A⁶) and for Pep A^1S show a symmetrical triple-banded electrophoretic pattern, which suggests that the activity attributable to Pep A⁵ or Pep A⁶ is about the same as that attributable to Pep A.^1S. Individuals presumed to be heterozygous for the rare allele Pep A⁵ or Pep A⁶ and for Pep A^1W show in red cells an asymmetrical electrophoretic pattern with a marked deficiency of activity attributable to Pep A^1W. In leucocytes, however, they show a symmetrical electrophoretic pattern indistinguishable from the patterns observed in individuals with the same rare allele in heterozygous combination with the common allele Pep A^S. 8. It is suggested that the wide variation in activity seen in red cells of individuals with the common electrophoretic phenotype Pep A1, is at least in part attributable to alleles (Pep A^1S and Pep A^1W) occurring at the same locus as the alleles which determine previously described electrophoretic variants. However, it remains possible that allelic differences at other gene loci and producing their effects in different ways may also contribute to the genetically determined variation in activity.