Modulated red blood cell survival by membrane protein clustering

Human and murine blood cells treated with ZnCl₂ and bis(sulfosuccinimidyl)suberate (BS³) (a cross linking agent) undergo band 3 clustering and binding of hemoglobin to red blood cell membrane proteins. These clusters induce autologous IgG binding and complement fixation, thus favouring the phagocytosis of ZnCl₂/BS³ treated cells by macrophages. The extension of red blood cell opsonization can be easily modulated by changing the ZnCl₂ concentration in the 0.1–1.0 mM range thus providing an effective way to affect blood cell recognition by macrophages. In fact, murine erythrocytes treated with increasing ZnCl₂ concentrations have proportionally reduced survivals when reinjected into the animal. Furthermore, the organ sequestration of ZnCl₂/BS³ treated cells strongly resembles the typical distribution of the senescent cells. Since the ZnCl₂/BS³ treatment can also be performed on red blood cells loaded with drugs or other substances, this procedure is an effective drug-targeting system to be used for the delivery of molecules to peritoneal, liver and spleen macrophages.     

Binding of vanadate to human erythrocyte ghosts and subsequent events

Spectroscopic techniques were used to investigate the interaction between vanadate and human erythrocyte ghosts. Direct evidence from ⁵¹V nuclear magnetic resonance (NMR) studies suggested that the monomeric and polymeric vanadate species may bind to the anion binding sites of band 3 protein of the erythrocyte membrane. The results of ⁵¹V NMR studies and the quenching effect of vanadate on the intrinsic fluorescence of the membrane proteins indicated that in the low concentration range of vanadate (<0.6 mm), monomeric vanadate binds mostly to the anion sites of band 3 protein with the dissociation constant close to 0.23 mm. The experiments of sulfhydryl content titration by the method of Ellman and residue sulfhydryl-labeled fluorescence spectroscopies clearly displayed that vanadate reacts directly with sulfhydryl groups. The appearance of the anisotropic election spin resonance (ESR) signal of vanadyl suggests that a small (c. 3%) amount of vanadate was reduced by sulfhydryl groups of membrane proteins. The fluidity and order of intact ghost membrane were reduced by the reaction with vanadate, as shown by the ESR studies employing the protein- and lipid-specific spin labels. It was concluded that although vanadates mainly bind to band 3 protein, a minor part of vanadate may oxidize the residue sulfhydryl groups of membrane proteins, and thus decrease the fluidity of erythrocyte membrane.     

Changes in DNA supertwist as a response of Bacillus subtilis towards different kinds of stress

Abstract The silent parD ( kis/kid ) stability operon of plasmid R1 is normally repressed by the co-ordinated action of the Kis and Kid proteins. In this report it is shown that a mutation in repA , the gene of the plasmid replication protein, that reduces two-fold the copy number of the plasmid, leads to the derepression of the parD system. This derepression can be prevented by a suppressor mutation in copB, a copy number control gene of plasmid R1, that increases the efficiency of replication of the repA mutant. Derepression of the wild-type parD system leads to high plasmid stability. These data show the activation of a plasmid stability operon by a mutation that reduces the efficiency of wild-type plasmid replication.     

Characterization of crystals of the thermostable DNA polymerase I from thermus aquaticus

Thermus aquaticus DNA polymerase I is an enzyme that is of both physiological and technological interest. It carries out template-directed polymerization of DNA at elevated temperatures and is widely used in polymerase chain reaction (PCR). We have obtained crystals of the enzyme that diffracts X-rays to at least 3.0 Å resolution in a cubic space group. Determination of the three-dimensional structure of the native enzyme along with those of relevant complexes will greatly enhance our knowledge of molecular events involved in DNA replication, will permit improvements in PCR, and will add to our knowledge of the structural bases of thermo stability in proteins. © 1995 Wiley-Liss, Inc.     

Establishing isostructural metal substitution in metalloproteins using 1H NMR, circular dichroism, and Fourier transform infrared spectroscopy.

Far-UV CD, 1H-NMR, and Fourier transform infrared (FTIR) spectroscopy are three of the most commonly used methods for the determination of protein secondary structure composition. These methods are compared and evaluated as a means of establishing isostructural metal substitution in metalloproteins, using the crystallographically defined rubredoxin from Desulfovibrio gigas and its well-characterized cadmium derivative as a model system. It is concluded that analysis of the FTIR spectrum of the protein amide I resonance represents the most facile and generally applicable method of determining whether the overall structure of a metalloprotein has been altered upon metal reconstitution. This technique requires relatively little biological material (ca. 300 micrograms total protein) and, unlike either CD or 1H-NMR spectroscopy, is unaffected by the presence of different metal ions, thus allowing the direct comparison of FTIR spectra before and after metal substitution.     

DNA topoisomerase activities in concanavalin A-stimulated lymphocytes

Topoisomerase activities have been measured in nuclear extracts of concanavalin A-stimulated lymphocytes. In parallel with the wave of DNA synthesis, type II topoisomerase activity was considerably increased. After 72 h treatment, this activity was stimulated approx. 20-fold over the activity in untreated cells. In contrast, type I topoisomerase was poorly stimulated after 24 h treatment, and 4-5-fold after 72 h. These findings, together with our previous results on regenerating rat liver, suggest a major role of topoisomerase II in DNA replication.     

Experimental studies on the function of the cortical cytoplasmic zone of the preprophase microtubule band

Summary Centrifugation of young seedlings ofTriticum durum andTriticum aestivum for 8–10 hours at 1,500–2,000 x g causes a serious disorder of the spatial organelle relationships in the interphase as well as the preprophase and mitotic subsidiary cell mother cells (SMCs). The nucleus, most organelles and cytoplasm are displaced to the centrifugal end of the cell, while the vacuoles lie at the other end. However, after centrifugation, the preprophase microtubule bands (PMBs) are nucleated and remain at the expected position close to the guard cell mother cells (GMCs). In some elongated SMCs the PMBs become completely separated from the nucleus. The mitotic spindle exhibits variable orientation and is usually formed at some distance from the PMB cortical zone.Cytokinesis in SMCs is spatially highly disturbed and the cell plate shows a variety of unpredictable dispositions, which seem to be determined by: 1. the position of the preprophase-prophase nucleus and the orientation of the mitotic spindle as well as their spatial relationships to the PMB cortical zone, and 2. the space available for cell plate growth. Many of the daughter cells exhibit a highly variable shape and size in different planes. Usually one edge of the cell plate partly or totally joins the anticlinal parent wall adjacent to the PMB cortical zone.In some SMCs ofZea mays andTriticum aestivum, the junction regions of the periclinal walls with the anticlinal ones, lined by the PMB cortical zone in normal SMCs, are detectably thickened after the arrest of mitosis and the prevention of interphase microtubule formation by a prolonged colchicine treatment. In a small number of protodermal cells of the same plants, participating in the development of stomatal complexes, irregular wall bodies or incomplete wall sheets were formed at wall regions lined by the PMB cortical zone.The presented observations are in line with the following hypotheses: 1. the PMB cortical zone interacts with the growing edges of the cell plate attracting it to fuse with the underlying parent wall when the latter approaches the former at a critical distance, and 2. in SMCs particular regions of the PMB cortical zone and/or the adjacent plasmalemma promote the local wall deposition in the absence of microtubules.     

ATP and the processivity of Xenopus laevis DNA polymerase α

We use specific restriction fragments as defined primers for DNA synthesis on single-stranded circular phage fd DNA. These structures are relatively poor templates for a highly purified DNA polymerase α from Xenopus laevis eggs. However, DNA synthesis is stimulated about 5-fold by addition of ATP to the reaction mixture. We show that the deoxynucleotide polymers, synthesized in the presence of ATP, are significantly longer than those produced in the absence of ATP. We also show that this effect is due to a more tenacious binding of DNA polymerase α to DNA and conclude that ATP increases the processivity of the enzyme.     

Transformation of Kluyveromyces lactis with the kanamycin (G418) resistance gene of Tn903

Direct selection of Kluyveromyces lactis resistant to the antibiotic G418 following transformation with the kanamycin resistance gene of Tn903 required the development of a procedure for producing high yields of viable spheroplasts and for the isolation of autonomous replication sequences (ARS). To obtain high yields of viable spheroplasts, cells were treated with (1) a thiol-reducing agent (L-cysteine), and (2) a high concentration of an osmotic stabilizer, 1.5 M sorbitol. Several ARS-containing plasmids were selected from a K. lactis recombinant DNA library in K. lactis and in Saccharomyces cerevisiae. Two of four ARS clones selected in K. lactis promoted transformation frequencies of 5-10 X 10(2) G418-resistant cells/micrograms of plasmid DNA. This frequency of transformation was at least twice as high as with ARS clones selected in S. cerevisiae. The stability of ARS-containing plasmids varied; after 20 generations of growth in the presence of G418, 16-38% of the cells remained resistant to the drug. In the absence of selection pressure less than 5% of the cells retained the drug-resistance phenotype. Plasmids containing the ARS1 or 2 mu replicon of S. cerevisiae failed to transform K. lactis for G418 resistance. Inclusion of S. cerevisiae centromere, CEN4, in a K. lactis ARS recombinant plasmid did not increase the stability of the plasmid in K. lactis, and marker genes on the vector segregated predominantly 4-:0+ through meiosis. We conclude that neither the ARS sequences or the centromere of S. cerevisiae was functioning in K. lactis.