Uncertainty in identification of blood group A subtypes by agglutination test

Identification of the blood group A subtypes, i.e. A1, A2, and A1-A2 intermediate (Aint), by agglutination test, particularly in AB red cells, is ambiguous. The expressions of A subtypes in red blood cells are the consequences of diverse formations of the A substances by the action of three types of blood group N-acetyl-galactosaminyl-transferases controlled by A1, A2, and Aint genes. Therefore, the A subtypes are more directly identified by examining the kinetic characters of A-enzymes existing in plasma. Several Black AB subjects classified as non-A1 by the agglutination test were identified as A1B and AintB on the enzyme basis. A subject serologically classified as A1 had A2-enzyme in her plasma, i.e. she is genetically A2O or A2A2. The present and previous studies indicate that red cell A2 status is occasionally expressed as a result of the combination of Aint and B, and of A1 and superactive B. The imbalance between A1/A2 and A1B/A2B observed in some Black populations could be attributed to high frequencies of the Aint and B. sup. genes in Blacks.     

Audiovisuals Reviews

Book reviewed in this article:
Vagrant Woman . A film by JOHN MARSHALL
A Forty Dollar Misunderstanding . A film by JOHN MARSHALL
Two Brothers . A film by JOHN MARSHALL
Wrong Kid . A film by JOHN MARSHALL
The Informant . A film by JOHN MARSHALL
After the Game . A film by JOHN MARSHALL
901/904 . A film by JOHN MARSHALL
An Investigation of a Hit and Run . A film by JOHN MARSHALL
A Legal Discussion of a Hit and Run . A film by JOHN MARSHALL
Nothing Hurt but My Pride . A film by JOHN MARSHALL
Youth and the Man of Property . A film by JOHN MARSHALL
Manifold Controversy . A film by JOHN MARSHALL
Appitsch and the Drunk . A film by JOHN MARSHALL
Henry is Drunk . A film by JOHN MARSHALL
T-Group . A film by JOHN MARSHALL
You Wasn't Loitering . A film by JOHN MARSHALL     

Ochratoxin production by Aspergillus species.

Ochratoxin production was tested in 172 strains representing species in sections Fumigati, Circumdati, Candidi, and Wentii of the genus Aspergillus by an immunochemical method using a monoclonal antibody preparation against ochratoxin A. Ochratoxin A was detected in Aspergillus ochraceus, A. alliaceus, A. sclerotiorum, A. sulphureus, A. albertensis, A. auricomus, and A. wentii strains. This is the first report of production of ochratoxins in the latter three species. Ochratoxin production by these species was confirmed by high-performance thin-layer chromatography and by high-performance liquid chromatography. The chemical methods also indicated the production of ochratoxin B by all of the Aspergillus strains mentioned above.     

Analysis of β3-endonexin mutants for their ability to interact with cyclin A

We have recently identified beta(3)-endonexin as a molecule that interacts with cyclin A-associated kinase. In this study, beta(3)-endonexin mutants were constructed by PCR-based site-directed mutagenesis, and characterized. Beta(3)-endonexin has a cyclin binding motif, RxL, in its N-terminal region, and two SP sequences which resemble a known target site for cyclin-dependent kinases (Cdks). The R5A/L7A mutant of beta(3)-endonexin, in which the RxL motif has been changed to AxA, is unable to bind to cyclin A, as revealed by two-hybrid experiments and in vitro pull-down assays. A GST-beta(3)-endonexin fusion, but not the corresponding R5A/L7A mutant, inhibits phosphorylation of Rb protein by cyclin A/Cdk2 in vitro. A cyclin A/Cdk2 kinase complex produced in, and purified from, insect cells phosphorylated GST-beta(3)-endonexin in vitro. The S33A or S46A mutant is partially phosphorylated by cyclin A/Cdk2, whereas no phosphorylation of the S33A/S46A double mutant is detectable. This demonstrates that these two serine residues, each of which is followed by a proline residue, are target sites for phosphorylation by cyclin A-associated kinase. The R5A/L7A mutant form of beta(3)-endonexin, which is defective for binding to cyclin A, is also not phosphorylated by cyclin A/Cdk2, confirming that the phosphorylation requires binding to cyclin A in the kinase complex. The neutralizing effect of beta(3)-endonexin on the toxicity associated with the expression of full-length human cyclin A in budding yeast is correlated with its ability to bind to cyclin A. Taken together, these data suggest that beta(3)-endonexin is phosphorylated by cyclinA/Cdk2 in vitro and that cyclin A-associated kinase activity is inhibited by the binding of beta(3)-endonexin to the kinase complex.     

Interaction amongst calcineurin subunits. Stimulatory and inhibitory effects of subunit B on calmodulin stimulation of subunit A phosphatase activity depend on Mn2+ exposure of the holoenzyme prior to its dissociation by urea

Calcineurin was dissociated into subunits A and B by 6 M urea in the presence (method A) and absence (method B) of MnCl2 and dissociated subunits were isolated by gel filtration in urea in the absence (method B) or presence (method A) of MnCl2. Phosphatase activity was associated with the A subunit isolated by either method. The phosphatase activity (nmol/mg) of subunit A isolated by method A was greater (2-5-fold) than by method B. Mn2+ increased subunit A phosphatase and calmodulin further increased the enzyme activity. Subunit B isolated by method A or B increased Mn2+ + calmodulin stimulated subunit A phosphatase prepared by method B but interestingly and unexpectedly inhibited such stimulated activity of the subunit A prepared by method A. These results imply the tightly bound cation (in our case, most likely Mn2+) with subunit A dramatically and differentially influences the effects of two Ca2+-binding proteins, calmodulin and subunit B, on the subunit A phosphatase.     

Mapping of sites on the surface membrane of mammalian cells

Summary Cells transformed by Simian Virus 40 have sites on the surface membrane for Concanavalin A (Con A) and a copolymer of ornithine, leucine (POL). The cells can be rapidly agglutinated by Con A, more slowly aggregated by POL, and they can be killed by both compounds. Treatment with Con A or POL has been used to select resistant cell variants from the transformed cells. Variants selected for resistance to Con A were also resistant to POL, but variants selected for resistance to POL were not resistant to Con A. The POL-selected variants showed less aggregation by POL but no decrease in agglutinability by Con A, whereas Con A-selected variants showed a decrease both in POL aggregation and Con A agglutination. The selection for both sites by Con A and only for POL sites by POL, can be explained in that the sites for POL are part of the sites for Con A and/or are included in clusters of Con A sites.Paper I in this series is Inbar, Ben-Bassat and Sachs (1971a).     

Microheterogeneity of molecular forms of arginase in mammalian tissues

Two isoforms of arginase, A1 and A2, were found in rat liver, submaxillary gland and kidney as well as beef kidney. In beef liver, however, A2 was the only detectable form. Two additional forms, A3 and A4, found only in rat kidney were probably artifactitious. A1 and A2 exhibited chromatographic and immunological microheterogeneity. While A1 in rat liver and submaxillary gland was excluded by DEAE-cellulose (pH 8.3) and retained on CM-cellulose (pH 7.5), that (A'1) in beef and rat kidneys was excluded by both ion-exchangers. A2 in all tissues was retained on DEAE-cellulose, but not on CM-cellulose. Both A1 and A2 in rat liver and beef kidney, A1 from rat submaxillary gland and A2 from beef liver were precipitated by antibodies to rat and beef liver arginases. None of the forms in rat kidney (A1, A2, A3 and A4) showed any cross-reactivity to either antibody. Rat submaxillary gland A2 was precipitated by anti-rat liver arginase, but activated by anti-beef liver arginase. While the major molecular forms were A1 in rat liver and submaxillary gland and A2 in beef liver and rat kidney, the two forms occurred in equal proportions in beef kidney. It appears that different isoforms might function as components of the urea cycle in the liver of different mammals and of the arginine catabolic pathway in different extrahepatic tissues.     

Occurrence of indole alkaloids among secondary metabolites of soil Aspergillus

The occurrence of indole alkaloids among secondary fungal metabolites was studied in species of the genus Aspergillus, isolated from soils that were sampled in various regions of Russia (a total of 102 isolates of the species A. niger, A. phoenicis, A. fumigatus, A. flavus, A. versicolor, A. ustus, A. clavatus, and A. ochraceus). Clavine alkaloids were represented by fumigaclavine, which was formed by A. fumigatus. alpha-Cyclopiazonic acid was formed by isolates of A. fumigatus, A. flavus, A. versicolor, A. phoenicis, and A. clavatus. The occurrence of indole-containing diketopiperazine alkaloids was documented for isolates of A. flavus, A. fumigatus, A. clavatus, and A. ochraceus. No indole-containing metabolites were found among the metabolites of A. ustus or A. niger.     

Identification of A chain cleavage sites in intact insulin produced by insulin protease and isolated hepatocytes

The degradation of insulin by the enzyme insulin protease and by isolated hepatocytes results in proteolytic cleavages in both the A and B chains of intact insulin. Previous studies have shown that one of the A chain cleavages is between A13 leucine and A14 tyrosine and that a second cleavage occurs carboxyl to the A14 residue. In the present study we have used insulin specifically iodinated on the A19 tyrosine and examined the A chain cleavages by the enzyme and by hepatocytes. Insulin degradation products were purified by HPLC and sequenced by automated Edman degradation. Only two A chain cleavage sites were identified, one the previously reported A13-A14 and the other between A14 tyrosine and A15 glutamine. These data thus identify the second A chain cleavage site and further support the role of insulin protease in hepatic metabolism of insulin.     

Phosphorylation of Saccharomyces cerevisiae choline kinase on Ser30 and Ser85 by protein kinase A regulates phosphatidylcholine synthesis by the CDP-choline pathway

The Saccharomyces cerevisiae CKI-encoded choline kinase is phosphorylated on a serine residue and stimulated by protein kinase A. We examined the hypothesis that amino acids Ser(30) and Ser(85) contained in a protein kinase A sequence motif in choline kinase are target sites for protein kinase A. The synthetic peptides SQRRHSLTRQ (V(max)/K(m) = 10.8 microm(-1) nmol min(-1) mg(-1)) and GPRRASATDV (V(max)/K(m) = 0.15 microm(-1) nmol min(-1) mg(-1)) containing the protein kinase A motif for Ser(30) and Ser(85), respectively, within the choline kinase protein were substrates for protein kinase A. Choline kinase with Ser(30) to Ala (S30A) and Ser(85) to Ala (S85A) mutations were constructed alone and in combination by site-directed mutagenesis and expressed in a cki1Delta eki1Delta double mutant that lacks choline kinase activity. The mutant enzymes were expressed normally, but the specific activity of choline kinase in cells expressing the S30A, S85A, and S30A,S85A mutant enzymes was reduced by 44, 8, and 60%, respectively, when compared with the control. In vivo labeling experiments showed that the extent of phosphorylation of the S30A, S85A, and S30A,S85A mutant enzymes was reduced by 70, 17, and 83%, respectively. Phosphorylation of the S30A, S85A, and S30A,S85A mutant enzymes by protein kinase A in vitro was reduced by 60, 7, and 96%, respectively, and peptide mapping analysis of the mutant enzymes confirmed the phosphorylation sites in the enzyme. The incorporation of (3)H-labeled choline into phosphocholine and phosphatidylcholine in cells bearing the S30A, S85A, and S30A,S85A mutant enzymes was reduced by 56, 27, and 81%, respectively, and by 58, 33, and 84%, respectively, when compared with control cells. These data supported the conclusion that phosphorylation of choline kinase on Ser(30) and Ser(85) by protein kinase A regulates PC synthesis by the CDP-choline pathway.     

Recombination and mutation of class II histocompatibility genes in wild mice

We have compared the tryptic peptide fingerprints of the A alpha, A beta, E alpha, and E beta subunits encoded by four wild-derived H-2 complexes expressing A molecules closely related to Ak. The A molecules encoded by these Ak-related mice have A alpha and A beta subunits that differ from A alpha k and A beta k by less than 10% of their tryptic peptides. Comparisons among the four wild-derived A molecules suggested that these contemporary A alpha and A beta alleles arose by sequential mutational events from common ancestor A alpha and A beta alleles. These results suggest that A alpha and A beta may co-evolve as an A beta A alpha gene duplex in wild mice. Tryptic peptide fingerprint comparisons of the E beta gene linked to these Ak-related A beta A alpha gene duplexes indicate that two encode E beta d-like subunits, whereas another encodes an E beta s-like subunit. These results strongly suggest that the A beta A alpha duplex and E beta recombine in wild mouse populations. The significantly different evolutionary patterns exhibited by the class II genes encoding A vs E molecules are discussed.     

Histone deacetylase HDAC2 silencing prevents endometriosis by activating the HNF4A/ARID1A axis

Endometriosis is the most major cause of chronic pelvic pain in women of reproductive age. Moreover, the involvement of histone deacetylase 2 (HDAC2) has been identified in endometriosis. However, the specific mechanism of HDAC2 remains to be further elusive. Therefore, this study was designed to explore the mechanism of HDAC2 orchestrating hepatocyte nuclear factor 4α/AT-rich interactive domain 1A (HNF4A/ARID1A) axis in endometriosis. Endometriosis cell line hEM15A and clinical endometriosis tissues were obtained, followed by gain- and loss-of-function assays in hEM15A cells. HDAC2, HNF4A and ARID1A expression was detected by immunohistochemistry and Western blot analysis. Cell viability was determined by Cell Counting Kit-8 Assay, invasion by Transwell assay and apoptosis by flow cytometry. HDAC2 enrichment in HNF4A promoter region and HNF4A enrichment in ARID1A promoter region was detected through chromatin immunoprecipitation. Mouse models of endometriosis were established, followed by immunohistochemistry of Ki-67 expression and TUNEL staining of apoptosis in ectopic tissues. HDAC2 was upregulated but HNF4A and ARID1A were downregulated in endometriosis tissues. HDAC2 inhibited HNF4A expression by deacetylation, and HNF4A was enriched in ARID1A promoter region to activate ARID1A. Silencing HDAC2 or overexpressing HNF4A or ARID1A diminished the viability and invasion and augmented the apoptosis of hEM15A cells. HDAC2 silencing reduced the area and weight of endometriosis tissues, suppressed endometriosis cell proliferation and accelerated endometriosis cell apoptosis. The inhibitory action of silencing HDAC2 via HNF4A/ARID1A axis was reproduced in mouse models. Collectively, HDAC2 silencing might upregulate HNF4A via repression of deacetylation to activate ARID1A, thus preventing the occurrence of endometriosis.     

Stimulation of leucine transport by a mitogen through intracellular Ca2+ increase in human peripheral lymphocytes

The effect of concanavalin A and ionophore A23187 on leucine uptake by human peripheral lymphocytes has been examined. Preincubation of the cells with 32 micrograms/ml concanavalin A or 0.1 microM A23187 increased leucine uptake by 67% and 100%, respectively. Both concanavalin A and A23187 could, within 2 min, induce a more than 2-fold increase in the cytoplasmic free Ca2+ concentration ([Ca2+]i). This increase by concanavalin A was completely blocked by the addition of 0.1 mM 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB-8) to incubation medium; TMB-8 partially blocked the action of A23187. The stimulation of leucine uptake by concanavalin A and A23187 was strongly inhibited by the presence of TMB-8 in the medium, whereas the basal uptake was not affected by this intracellular Ca2+ antagonist. Amiloride did not inhibit the stimulation of leucine uptake by concanavalin A. The concanavalin A- and A23187-induced elevation of [Ca2+]i was accompanied by membrane hyperpolarization. Concanavalin A-stimulated leucine uptake was greatly inhibited by the presence of an excess of 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid. These results indicate that the increase in [Ca2+]i may function as a signal of the stimulation by mitogen of leucine uptake mediated by system L, finally inducing membrane hyperpolarization in human lymphocyte.     

Essential role of cAMP-response element-binding protein activation by A2A adenosine receptors in rescuing the nerve growth factor-induced neurite outgrowth impaired by blockage of the MAPK cascade

We found in the present study that stimulation of the A(2A) adenosine receptor (A(2A)-R) using an A(2A)-selective agonist (CGS21680) rescued the blockage of nerve growth factor (NGF)-induced neurite outgrowth when the NGF-evoked MAPK cascade was suppressed by an MEK inhibitor (PD98059) or by a dominant-negative MAPK mutant (dnMAPK). This action of A(2A)-R (designated as the A(2A)-rescue effect) can be blocked by two inhibitors of protein kinase A (PKA) and was absent in a PKA-deficient PC12 variant. Activation of the cAMP/PKA pathway by forskolin exerted the same effect as that by A(2A)-R stimulation. PKA, thus, appears to mediate the A(2A)-rescue effect. Results from cAMP-response element-binding protein (CREB) phosphorylation at serine 133, trans-reporting assays, and overexpression of two dominant-negative CREB mutants revealed that A(2A)-R stimulation led to activation of CREB in a PKA-dependent manner and subsequently reversed the damage of NGF-evoked neurite outgrowth by PD98059 or dnMAPK. Expression of an active mutant of CREB readily rescued the NGF-induced neurite outgrowth impaired by dnMAPK, further strengthening the importance of CREB in the NGF-mediated neurite outgrowth process. Moreover, simultaneous activation of the A(2A)-R/PKA/CREB-mediated and the phosphatidylinositol 3-kinase pathways caused neurite outgrowth that was not suppressed by a selective inhibitor of TrkA, indicating that transactivation of TrkA was not involved. Collectively, CREB functions in conjunction with the phosphatidylinositol 3-kinase pathway to mediate the neurite outgrowth process in PC12 cells.     

Glycogen synthase sensitivity to glucose-6-P is important for controlling glycogen accumulation in Saccharomyces cerevisiae

Glycogen is a storage form of glucose utilized as an energy reserve by many organisms. Glycogen synthase, which is essential for synthesizing this glucose polymer, is regulated by both covalent phosphorylation and the concentration of glucose-6-P. With the yeast glycogen synthase Gsy2p, we recently identified two mutants, R579A/R580A/R582A [corrected] and R586A/R588A/R591A, in which multiple arginine residues were mutated to alanine that were completely insensitive to activation by glucose-6-P in vitro (Pederson, B. A., Cheng, C., Wilson, W. A., and Roach, P. J. (2000) J. Biol. Chem. 275, 27753-27761). We report here the expression of these mutants in Saccharomyces cerevisiae and, as expected from our findings in vitro, they were not activated by glucose-6-P. The R579A/R580A/R582A [corrected] mutant, which is also resistant to inhibition by phosphorylation, caused hyperaccumulation of glycogen. In contrast, the mutant R586A/R588A/R591A, which retains the ability to be inactivated by phosphorylation, resulted in lower glycogen accumulation when compared with wild-type cells. When intracellular glucose-6-P levels were increased by mutating the PFK2 gene, glycogen storage due to the wild-type enzyme was increased, whereas that associated with R579A/R580A/R582A [corrected] was not greatly changed. This is the first direct demonstration that activation of glycogen synthase by glucose-6-P in vivo is necessary for normal glycogen accumulation.     

Phosphorylation of amyloid-β peptide at serine 8 attenuates its clearance via insulin-degrading and angiotensin-converting enzymes

Accumulation of amyloid-β peptides (Aβ) in the brain is a common pathological feature of Alzheimer disease (AD). Aggregates of Aβ are neurotoxic and appear to be critically involved in the neurodegeneration during AD pathogenesis. Accumulation of Aβ could be caused by increased production, as indicated by several mutations in the amyloid precursor protein or the γ-secretase components presenilin-1 and presenilin-2 that cause familial early-onset AD. However, recent data also indicate a decreased clearance rate of Aβ in AD brains. We recently demonstrated that Aβ undergoes phosphorylation by extracellular or cell surface-localized protein kinase A, leading to increased aggregation. Here, we provide evidence that phosphorylation of monomeric Aβ at Ser-8 also decreases its clearance by microglial cells. By using mass spectrometry, we demonstrate that phosphorylation at Ser-8 inhibited the proteolytic degradation of monomeric Aβ by the insulin-degrading enzyme, a major Aβ-degrading enzyme released from microglial cells. Phosphorylation also decreased the degradation of Aβ by the angiotensin-converting enzyme. In contrast, Aβ degradation by plasmin was largely unaffected by phosphorylation. Thus, phosphorylation of Aβ could play a dual role in Aβ metabolism. It decreases its proteolytic clearance and also promotes its aggregation. The inhibition of extracellular Aβ phosphorylation, stimulation of protease expression and/or their proteolytic activity could be explored to promote Aβ degradation in AD therapy or prevention.     

巨噬细胞吞噬过程中膜磷脂流动性和受体流动性的关系

本文用ＦＲＡＰ（ｆｌｕｏｒｅｓｃｅｎｃｅｒｅｃｏｖｅｒｙａｆｔｅｒｐｈｏｔｏｂｌｅａｃｈｉｎｇ）技术,测量了静息状态和刀豆素Ａ刺激不同时间后巨噬细胞膜磷脂、ＣｏｎＡ受体扩散系数和荧光恢复率的变化。结果显示ＣｏｎＡ刺激后膜磷脂和ＣｏｎＡ受体的扩散系数和荧光恢复率均较静息状态的巨噬细胞明显降低,磷脂流动性的变化与ＣｏｎＡ受体流动性的变化呈正相关。提示受体介导内吞导致的膜磷脂流动性的降低,可能是由于配体与细胞膜上受体结合形成配体－受体复合体,增加了受体的负荷,使受体的流动性降低,进而使膜磷脂的流动性降低。巨噬细胞内吞过程中膜磷脂和ＣｏｎＡ受体流动性的降低,可能还与ＣｏｎＡ刺激后巨噬细胞胞浆ｐＨ值有关。