Zur Populationsgenetik der sauren Phosphatase der Erythrocyten (EC: 3.1.3.1): Phänotypen- und Allelhäufigkeiten in der ČSSR

Summary Within a population sample of 307 blood donors of Prague the polymorphism of the red cell acid phosphatase has been investigated. Gene frequency estimates are: P^a=0.365, P^b=0.578, P^c=0.057.     

The rare silent gene Po of human red cell acid phosphatase in a second family in poland

The authors describe a Polish family (three generations) with a silent gene P^o of human red cell acid phosphatase. The phosphatase levels of 4 family members were about half the normal value.     

Quantitative evidence of a silent gene P° of human red cell acid phosphatase in south Polish population

The distribution of red cell acid phosphatase types in 3244 unrelated Polish adults is reported. Gene frequencies P^a = 0.3594, P^b = 0.5643 and P^c = 0.0763 were obtained. In a forensic case of disputed paternity an apparent mother/child incompatibility respect to red cell acid phosphatase was found, the mother appearing as type B and the child as type A. Determination of acid phosphatase activity suggested the presence of a silent gene P°. The phosphatase levels were about half the values expected, as determined in 237 adults representing the different phenotypes.     

Zur Populationsgenetik der sauren Phosphatase der Erythrocyten (E C 3.1.3.2): Phänotypen-und Allelhäufigkeiten in Südwestdeutschland

Summary Within an population sample of 300 individuals of Southwestern Germany the red cell acid phosphatase polymorphism is investigated. Gene frequency estimates are: P^a=0.31, P^b=0.643, P^c=0.047.

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Direktor: Prof. Dr. med. Dr. H. Baitsch

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Human red cell acid phosphatase polymorphism

Red cell acid phosphatase phenotypes were determined in 401 unrelated persons from Southwestern Germany. The frequencies of genes P^a, P^b and P^c were estimated to be p=0.328, q=0.630 and r=0.042.Experiences in 101 cases of disputed paternity with 151 men involved are reported. 22 men could be excluded from paternity on the basis of red cell acid phosphatase phenotypes.In all 140 mother-child-combinations tested the distribution of red cell acid phosphatase variants was compatible with the genetic model suggested by Hopkinson et al.Usefulness of the system in forensic cases of disputed paternity is discussed.Data contained in this paper will also constitute part of the thesis of cand. med. Karl-Henning Lichte.     

Polymorphism of the red cell acid phosphatase in the Swiss population

Summary The distribution of the red cell acid phosphatase groups was studied on 1365 blood samples of Swiss individuals. The distribution is in agreement with the Hardy-Weinberg equilibrium. Gene frequencies similar to those observed in other Caucasian populations were obtained (P^A=0.345, P^B=0.607, P^C=0.049). In 331 mother/child-combinations, we found no theoretically impossible combinations.     

An examination of the age-related patterns of decay of acid phosphatase (ACP1) in human red cells from individuals of different phenotypes

A study has been made of the decay of acid phosphatase (ACP₁) in the human red cell using red cell fractions of different mean ages prepared by density gradient centrifugation. Red cells from acid phosphatase type A and type B individuals were used in the study. Acid phosphatase activity of the red cell fractions was determined by two different assay methods. The results obtained were comparable and have been combined. Acid phosphatase type A and type B showed a biphasic decay pattern with a rapid early loss of activity, followed by a more gradual rate of decline. Type A appeared to decay more rapidly than type B in both decay phases. It is proposed that differences in stability between type A and type B in vivo may explain the observed differences in activity between the enzyme types. There was no evidence for the generation of secondary isozymes by acid phosphatase type A or type B during red cell aging.     

ATPase and phosphatase activities from human red cell membranes: II. The effects of phospholipases on Ca2+-dependent enzymic activities

Summary Treatment of human red cell membranes with pure phospholipase A₂ results in a progressive inactivation of both Ca²⁺-dependent and (Ca²⁺+K⁺)-dependent ATPase and phosphatase activities. When phospholipase C replaces phospholipase A₂, Ca²⁺-dependent ATPase activity and Ca²⁺-dependent phosphorylation of red cell membranes are lost, while Ca²⁺-dependent phosphatase activity is enhanced and its apparent affinity for Ca²⁺ is increased about 20-fold. Activation of Ca²⁺-dependent phosphatase following phospholipase C treatment was not observed in sarcoplasmic reticulum preparation. Phospholipase C increases the sensitivity of the phosphatase to N-ethylmaleimide but has little effect on the kinetic parameters relating the phosphatase activity to substrate and cofactors, suggesting that no extensive structural disarrangement of the Ca²⁺-ATPase system has occurred after incubation with phospholipase C.     

The purification and kinetic properties of biophosphoglycerate synthase from horse red blood cells.

Bisphosphoglycerate synthase from horse red cells has been purified to apparent homogeneity by a simple and efficient new procedure incorporating chromatography on a column of Sepharose 4B derivatized with blue dextran. The enzyme is similar to the human red cell synthase in subunit size. It is phosphorylated by either glycerate-1,3-P₂ or glycerate-2,3-P₂ to form a phosphoenzyme with the acid-lability of a histidyl phosphate. In addition to the synthase activity (glycerate-1,3-P₂ → glycerate-2,3-P₂), k_cat 12.5 s^?1, the enzyme has bisphosphoglycerate phosphatase activity in the presence of glycolate-2-P (glycerate-2,3-P₂ → glycerate-P + P_i), k_cat 2.6 s^?1 and phosphoglycerate mutase activity (3-PGA ? 2-PGA), k_cat 1.7 s^?1. The energy of activation for the synthase reaction is 9.38 kcal/mol. Lineweaver-Burk plots of the kinetic data are parallel lines. In contrast intersecting patterns were obtained from similar experiments done with the human red cell enzyme. Further investigation is required to explain these differences. This enzyme may function as both synthase and phosphatase for bisphosphoglycerate in the red blood cell.     

Maternal and zygotic requirement for thepolyhomeotic complex genetic locus inDrosophila

Summary The complex genetic locuspolyhomeotic (ph) is a member of thePolycomb (Pc)-group of genes and as such is required for the normal expression of ANT-C and BX-C genes. It also has probably other functions since amorphicph alleles display a cell death phenotype in the ventral epidermis of 12-h-old embryos. Here it is shown that lethal alleles ofph (amorph and strong hypomorph) show transformation of most of their segments towards AB8. Theph ⁺ product is required autonomously in imaginal cells. The total lack ofph ⁺ function prevents viability of the cuticular derivatives of these cells.ph has a strong maternal effect on segmental identity and epidermal development that can not be rescued by one paternally supplied dose ofph ⁺ in the zygote. These phenotypes differ substantially from those of previously describedPc-group genes. AmongPc-group genes,ph seems to be the only one that is strongly required both maternally and zygotically for normal embryonic development.     

Cell cycle defects in polyhomeotic mutants are caused by abrogation of the DNA damage checkpoint

Polycomb group (PcG) genes are required for heritable silencing of target genes. Many PcG mutants have chromatin bridges and other mitotic defects in early embryos. These phenotypes can arise from defects in S phase or mitosis, so the phenotype does not show when PcG proteins act in cell cycle regulation. We analyzed the cell cycle role of the proximal subunit of Polyhomeotic (PhP) in Drosophila. Time-lapse imaging reveals that chromatin bridges formed during mitosis are able to resolve but sometimes result in chromosome breakage. Chromosome bridging is also observed in canonical cell cycles occurring in larval brains and is therefore not unique to the rapid embryonic cycles. PhP colocalizes with chromatin in S phase but not in mitosis in early embryos, indicating a direct role in DNA synthesis. Time lapse imaging of ph^p mutants reveals an acceleration of S phase, showing that ph^p regulates S phase length. Like ph^p mutations, mutations in DNA damage checkpoints result in S phase acceleration. Consistent with this model, mutations in ph do not affect DNA synthesis rates, but exhibit impaired ability to block cell cycle progression following exposure to gamma-rays. Our data show that the mitotic defects of ph^p are caused by defects in the DNA damage response that occurs after DNA replication in S phase, and we propose that PhP has a direct role in DNA damage repair.     

Sequencing, cloning, and expression of human red cell-type acid phosphatase, a cytoplasmic phosphotyrosyl protein phosphatase.

Low molecular weight phosphotyrosyl protein phosphatases of human placenta and human red cell were purified and sequenced by a combination of Edman degradation and tandem mass spectrometry. Screening of a human placental lambda gt11 cDNA library yielded overlapping cDNA clones coding for two distinct human cytoplasmic low molecular weight phosphotyrosyl protein phosphatases (HCPTPs). The two longest clones, designated HCPTP1-1 and HCPTP2-1, were found to have identical nucleotide sequences, with the exception of a 108-base pair segment in the middle of the open reading frame. Polymerase chain reaction studies with human genomic DNA suggest that the difference between HCPTP1-1 and HCPTP2-1 does not result from alternative RNA splicing. Studies with a human chromosome 2-specific library confirmed that these sequences are located on chromosome 2, which is known to be the location of red cell acid phosphatase locus ACP1. The coding sequences of HCPTP1-1 and HCPTP2-1 were placed downstream from a bacteriophage T7 promoter and the proteins were expressed in Escherichia coli. The resulting recombinant enzymes (designated HCPTP-A and HCPTP-B, respectively) showed molecular weights of 18,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and both of them exhibited immunoreactivity with antisera raised against authentic human placental and bovine heart enzymes. The expressed proteins were highly active towards the phosphatase substrates p-nitrophenyl phosphate, beta-naphthyl phosphate, and O-phospho-L-tyrosine, but not alpha-naphthyl phosphate, threonine phosphate, or O-phospho-L-serine. HCPTP-A and -B possessed effectively identical amino acid compositions, immunoreactivities, inhibition by formaldehyde, and kinetic properties when compared with two human red cell acid phosphatase isoenzymes. It is concluded that HCPTP-A and -B are the fast and slow forms of red cell acid phosphatase, respectively, and that this enzyme is not unique to the red cell but is instead expressed in all human tissues.     

Potassium-activated phosphatase from human red blood cells

Summary The cell membrane K⁺-activated phosphatase activity was measured in reconstituted ghosts of human red cells having different ionic contents and incubated in solutions of varying ionic composition. When K⁺-free ghosts are suspended in K⁺-rich media, full activation of the phosphatase is obtained. Conversely, very little ouabainsensitive activity is detected in K⁺-rich ghosts suspended in K⁺-free media. These results, together with the fact that Na⁺ competitively inhibits the effects of K⁺ only when present externally, show that the K⁺ site of the membrane phosphatase is located at the outer surface of the cell membrane. The Mg⁺⁺ requirements for K⁺ activation of the membrane phosphatase are fulfilled by internal Mg⁺⁺. Addition of intracellular Na⁺ to ATP-containing ghosts raises the apparent affinity of the enzyme for K⁺, suggesting that the sites where ATP and Na⁺ produce this effect are located at the inner surface of the cell membrane. The asymmetrical features of the membrane phosphatase are those expected from the proposed role of this enzyme in the Na⁺–K⁺-ATPase system.The authors are established investigators of the Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina.     

Red cell isozymes in the Eastern Carolines

Four populations of islanders (Ponapeans, Mokilese, Pingelapese, and Kusaieans) in the Eastern Caroline Islands have been surveyed for variability in red cell acid phosphatase, phosphoglucomutase, 6-phosphogluconate dehydrogenase, adenylate kinase and glucose-6-phosphate dehydrogenase. The following gene frequencies were observed: P^a = 0.0904, PGM²₁ = 0.1015, and PGD^B = 0.0259. No genetic variation was encountered in the AK and G6PD systems.     

Polyhomeotic: A gene of Drosophila melanogaster required for correct expression of segmental identity

Summary A new locus in Drosophila melanogaster that is required for the correct expression of segmental identity has been discovered. The new locus, termed polyhomeotic (ph), is X-linked and maps cytologically to bands 2D2-3. Homozygous ph flies have homeotic transformations similar to those of known dominant gain of function mutants in the Antennapedia and bithorax complexes (ANT-C, BX-C), and in addition show loss of the humerus. ph interacts with three other similar mutations: Polycomb (Pc), Polycomblike (Pcl), and extra sex comb (esc), and acts as a dominant enhancer of Pc. The expression of ph depends on the ANT-C and BX-C dosage. ph has no embryonic phenotype, but temperature shift studies on ph ² show that the ph ⁺ product is required during embryogenesis and larval development. We propose that ph mutants in some way disrupt the normal expression of the ANT-C and BX-C, and, therefore, that ph ⁺ is needed for maintenance of segmental identity.     

Methodische Studien zur Cytochemie der Leukozytenphosphatasen

Zusammenfassung Es werden mehrere Techniken angeführt, die für den Phosphatasenachweis an Ausstrich- und Tupfpräparaten entwickelt wurden und hier sicher reproduzierbare Ergebnisse liefern. Im Prinzip handelt es sich um die Gomori-Technik des Nachweises der sauren Phosphatase (Bleisulfid-Methode). Benutzt wurden folgende Substrate: 1. Glycerophosphat beiph 6,6, 2. Aneurinpyrophosphat beiph 7,2, 3. Adenosin-3-monophosphorsäure beiph 6,0, 4. Adenosin-5-monophosphorsäure beiph 6,0. Die Inkubationszeit betrug 2–3 Std bei 37° C. Bei allen Substraten konnte die Reaktion durch vorhergehende Cyanidbehandlung komplett gehemmt werden.Auf die Reaktion der verschiedenen Blutzellen wird hingewiesen. Die starke Aktivität der Kapillaren und Retikulumzellen wird ausdrücklich betont. Die angeführten Methoden erscheinen besonders geeignet zum Studium der Retikulumzellen in bioptischen Knochenmarkspunktaten.

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Summary Several cytochemical techniques are described demonstrating the activity of phosphatases in blood and bone marrow smears. All theses methods are a modification of the lead-sulfide principle ofGomori for the demonstration of acid phosphatase. The following substrates were being used: 1. Sodium--Naphthylphosphate,ph 9,2. 2. Aneurinpyrophosphate (Cocarboxylase),ph 7,2. 3. Adenosine-3-monophosphoric acid,ph 6,0. 4. Adenosine-5-monophosphoric acid,ph 6,0. 5. Sodium-glycerophosphate,ph 6,6. The incubation time was 2–3 hours at 37° C. With all substrates the reaction was completely inhibited by preceding incubation in a 0,1 m cyanide solution.The reaction of different bone marrow cells is shortly described. The very strong activity of capillaries and reticulum cells is pointed out. The methods described seem especially useful for the study of reticulum cells in bone marrow smears.

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Mit 6 Textabbildungen     

Mmebrane phosphatase and active transport in red cells from different species

The K⁺-dependent phosphatase activity from red cell membranes from different mammalian species shows a close relationship with both the rate of active potassium influx and (Na⁺ + K⁺)-dependent ATPase activity. This finding supports the view that membrane phosphatase activity is related to the cation transport system.     

Influence of bio-optical parameter variability on the reflectance peak position in the red band of algal bloom waters

On the basis of field measurements, the quantitatively different relationships of peak position in the red band of the remote sensing reflectance vs. Chl concentration are found in the bloom waters of the diatom Skeletonema costatum and the dinoflagellate Prorocentrum donghaiense in coastal areas of the East China Sea. Model simulations of remote sensing reflectance, R_rs, accounting for the influence of variations in the bio-optical parameters such as chlorophyll fluorescence quantum efficiency, Φ, and specific absorption coefficient, a_ph^⁎, are carried out to analyze the characteristics of this spectral peak. The strong effect of fluorescence on the magnitude of R_rs results in the inhibition of the shift of the peak to longer wavelengths, increasing Φ enhances this effect. Increasing a_ph^⁎, specifically in the red-wavelength band, causes a sharper shift in the red peak position by decreasing the effect of the fluorescence. The dominant parameter governing the slope of the shift is a_ph^⁎. The analysis indicates that the higher a_ph^⁎ of S. costatum in the red region is primarily responsible for the much higher slope of the peak shift than for that of P. donghaiense. We show that the relationship between the peak position and Chl concentration may be useful for discriminating S. costatum blooms from those due to P. donghaiense, although information about chlorophyll fluorescence quantum efficiency should be included. Finally, we show that using the band ratio R_rs(708 nm)/R_rs(665 nm) instead of Chl in the relationship with peak position can be useful for the practical identification of S. costatum blooms from hyperspectral measurements of remote sensing reflectance.     

Phosphatase activity in cultured glioma and neuroblastoma cells

Acid phosphatase activity in human glioma cells (138 MG) and mouse neuroblastoma cells (C 1300) was associated with structures accumulating neutral red and acridine orange. Only neuroblastoma cells gave a significant positive histochemical reaction for alkaline phosphatase. Glioma and neuroblastoma cell homogenates exhibited maximal phosphatase activity at pH 5 as measured by spectrophotometer. The specific activity; μmoles phosphate released per hour/mg protein was 1.1 in glioma and 0.9 in neuroblastoma. At pH 8, glioma cells lacked activity whereas neuroblastoma cells showed another maximum. The acid phosphatase activity of both cell types was strongly inhibited by CuCl₂ (0.3 mM) and NaF (10 mM) and moderately by -tartaric acid (10 mM). cGMP (1 mM) stimulated the phosphatase activity of both cell lines. db-cAMP, in serum-free medium, induced characteristic morphological changes of the cells studied. This process was unaffected by CuCl₂, c-GMP and -tartaric acid. db-cAMP (1 mM) inhibited proliferation in both glioma and neuroblastoma cells during a 48 h incubation in serum-containing medium. This growth inhibition was associated with an increase in acid phosphatase activity of the glioma but not of the neuroblastoma cells.