Biochemical studies on abnormal erythrocyte membranes. Protein abnormality of erythrocyte membrane in biliary obstruction

Biochemical studies on erythrocyte membranes from eleven obstructive jaundice patients (due to various disorders) have been undertaken, By scanning electron microscopic observation these erythrocytes were spur and target in appearance. The lipid composition showed a marked increase in both cholesterol and phosphatidylcholine. In addition to these changes, it was unexpectedly demonstrated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate that a specific membrane protein component 4.2 was reduced or absent in all cases tested. This membrane protein abnormality was identical with that of hereditary spherocytosis erythrocyte membranes. It is of particular interest to note that after surgical relief of biliary obstruction in a typical case of common duct cholelithiasis, the disc electrophoretic pattern of erythrocyte membranes became normal and both lipid composition and red cell morphology returned to normal.     

Chemical characterization and distribution of ABO blood group active glycoprotein in human erythrocyte membrane

Occurrence and distribution of glycoprotein H substances in blood type O human erythrocyte membrance were studied using an alpha N-acetylgalactosaminyltransferase (A-enzyme) purified from milk of blood type A women. Erythrocytes of blood group O, nonsecretors and secretors alike, contain H substances that serve as a substrate for this transferase, and the H determinants seem to occur in all three erythrocyte membrane glycoproteins (periodic acid-Schiff (PAS)-1, PAS-2, and PAS-3). Structural studies of the A glycoproteins obtained by the transferase action, suggested that all erythrocyte H glycoproteins contain only type 2 antigenic determinant.     

Mass spectrometric analysis of 2-deoxyribonucleoside and 2'-deoxyribonucleotide adducts with aldehydes derived from lipid peroxidation

An important emerging issue in chemical carcinogenesis is the role that products of endogenous metabolism play in formation of covalently modified DNA. One example is the formation of alpha, beta-unsaturated aldehydes as a result of endogenous and drug-stimulated lipid peroxidation. Malondialdehyde (MDA), crotonaldehyde (CR), 2-hexenal (HX), and 4-hydroxy-2-nonenal (HNE) react covalently with 2'-deoxyguanosine (dG) and 2'-deoxyadenosine (dA) residues on DNA to form promutagenic cyclic adducts that may be important in the etiology of cancer in humans and animals. The accurate quantification of such adducts provides a powerful tool in molecular epidemiology for assessing carcinogenic risks from various lifestyle choices (e.g. diet, drug use) in humans. 32P-Postlabeling is recognized as one of the most sensitive methods available for detection of DNA adducts in human tissues, but without adequate validation such methodology can yield inaccurate quantitative measurements. We have used LC separations in conjunction with electrospray ionization MS and tandem MS (triple quadrupole and hybrid quadrupole-orthogonal acceleration time of flight analyzers) to characterize MDA-, CR-, HX- and HNE-modified dG and nucleotide (3'- and 5'-monophosphate; 3',5'-bisphosphate) adducts. These data have been used to validate 32P-postlabeling methods for quantification of low level MDA-dG adducts formed in DNA of human and animal tissues. Availability of reliable methods for quantification of endogenous DNA damage in humans and animals is essential for determining unknown etiologies of cancer and for the assessment of cancer risks in humans.     

Protective effect of active components extracted from radix Astragali on human erythrocyte membrane damages caused by reactive oxygen species

A study has been made on the protective activities of active components extracted from Radix Astragali by using reactive oxygen species initiated lipid peroxidation of purified human erythrocyte membrane. The results show that the total flavonoids of Astragalus and total saponins of Astragalus can significantly inhibit the membrane lipid peroxidation generated by O2.-, H2O2 and UV rays, while the total polysaccharide of Astragalus possesses weaker protective activity.     

Nonmediated flip-flop of anionic phospholipids and long-chain amphiphiles in the erythrocyte membrane depends on membrane potential

The nonmediated inward translocation (flip) of the anionic fluorescent N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)- (NBD-)labeled phospholipid phosphatidylmethanol (PM) from the outer to the inner membrane leaflet of human erythrocytes and vice versa depends on membrane potential. Interestingly, inside-positive potentials due to chloride gradients and the native chloride conductance of the cells resulted in an increase of the flip rates. This flip enhancement could be suppressed by addition of gramicidin D, which increases cation conductance, or 4,4'-diisothiocyanatostilbene-2,2'-disufonate (DIDS), which inhibits anion conductance. Conversely, inside negative potentials established by an outward-directed K+ gradient in the presence of gramicidin on DIDS-treated cells resulted in a decrease of flip rate. Flip rate exhibited an exponential dependence on membrane potential. The opposite effects of the positive and negative potentials were obtained for the outward translocation (flop) from the inner to the outer membrane leaflet. Similar potential dependencies were found for the nonmediated flip of anionic NBD-labeled phosphatidic acid (PA) and 2-(N-decyl)aminonaphthalene-6-sulfonic acid (2,6-DENSA) following blockage of the band-3-mediated component of flip. The membrane potential also influences the stationary distribution of the anionic lipids between the inner and outer leaflets. The distribution is shifted to the inner leaflet by increasingly positive potentials and to the outer leaflet by increasingly negative potentials. It is concluded that nonmediated flip-flop of the anionic phospholipids and the long-chain sulfonate represents electrogenic translocation of the unprotonated charged lipids across the hydrophobic barrier.     

不同脱氧程度锰钢耐点蚀性能比较

选择四种不同脱氧程度的锰钢,在pH10的3%(质量分数)NaCl溶液中进行动电位极化实验,比较钢的点蚀诱发敏感性;在3%(质量分数)海盐水中进行室内间浸挂片实验,评价钢的点蚀扩展速度;利用扫描电镜(SEM)和电子探针(EPMA)分析了钢中夹杂物组成和腐蚀形貌.结果表明,随着脱氧程度的加强,钢中的硫化物夹杂由短棒状变为长条状.脱氧程度越差,钢基体热力学稳定性越强,钢的点蚀诱发敏感性也越弱.室内间浸挂片实验结果表明,脱氧程度较强的锰钢表现出更强的点蚀扩展能力.     

Comparative studies on different small intestinal oligosaccharidase activities in some vertebrates and invertebrates like molluscs

Oligosaccharidase activities of the small intestinal mucosal homogenates were measured in vertebrates viz fish, toad, garden-lizard (calotes), pigeon, rat and some invertebrates viz, molluscsa. Maximum activities of the enzymes Lactase, Sucrase and Maltase were found in the mammalian species rat, whereas much less activities were found in the non-mammalian vertebrates among which toad shows the highest values and garden lizard the lowest. Among the invertebrates Pila globosa shows higher values of all the enzymes than Achatina fulica. The results obtained have been discussed in the lights of phylogeny and diet habits.     

Chemistry of loteprednol etabonate and related steroids. II. Reactions at ring C and NMR structural studies of the resulting compounds

The effects of three inducers of differentiation, phorbol myristate acetate (PMA), retinoic acid (RA) and interferon-gamma (IFN-gamma), on the temporal regulation of vitamin D receptor (VDR) expression in HL-60 cells were analyzed by Northern blotting and immunofluorescence assays. VDR, at the protein level, expressed by 81% of uninduced cells, was reduced to 57% after 48 h of PMA or 96 h of RA treatment, preceded by growth inhibition and cell differentiation, evaluated by CD11b expression. Sorted CD11b positive cells in G0/G1 phase exhibited 53% the VDR content of CD11b negative cells (distributed throughout the cell cycle). PMA also induced an increase in PKC beta and PKC alpha mRNA and protein. Simultaneous exposure to PMA and sphingosine blocked stimulation of CD11b and PKC expression without affecting growth arrest and VDR down regulation. Similar effects were observed during sphingosine treatment. In IFN-gamma differentiated cells, the proportion of cells in G0/G1 phase was unchanged and VDR protein was unaltered as compared to uninduced cells. Control cells in G0/G1 expressed less VDR than cells in S and G2/M phases (74% and 59% respectively). All results suggest that in HL-60 cells, reduction of VDR expression is related to growth inhibition rather than to the differentiation process.     

The effects of mitomycin-C and temperature on dynamical properties of human erythrocyte membrane

We investigated the mode of action of a new quinoline derivative, TAK-603 (ethyl 4-(3,4-dimethoxyphenyl)-6,7-dimethoxy-2-(1,2,4-triazol-1-ylmeth yl) quinoline-3-carboxylate), in adjuvant arthritis (AA), a model of rheumatoid arthritis. AA rat splenocytes transferred the arthritis to normal syngeneic rats upon inoculation, but the cells from AA rats treated with TAK-603 (6.25 mg/kg/day) caused only mild arthritis with significantly less foot pad swelling and a lower arthritis score. An effect of TAK-603 in the induction phase of AA was suggested. TAK-603 had little effect on CD4+ and CD8+ T-cell populations in the AA rat splenocytes. We therefore estimated the frequency of T-cells which are reactive to the so-called disease causative antigen using a limiting dilution assay (LDA). The ratio of T-cells responsive to PPD, which increased in AA rat splenocytes with the severity of the arthritis, was reduced in AA rats treated with TAK-603. Furthermore, the ratio of MBP (myelin basic protein)-reactive T-cells, which were generated in experimental allergic encephalomyelitis (EAE) rats, were also reduced by TAK-603 administration. These data suggest that TAK-603 acts on the immune system and reduces the number of cells reactive to the relevant antigen.     

Comparative analysis of one-particle mathematical models in materials science and oncology. II. Qualitative study of the equations

A system of two nonlinear first-order differential equations is analyzed that describe the dynamics of the interaction between lymphocytes and tumor cells in the body. The model utilized takes into account the effect of a magnetic field. A concept is formulated concerning the analogy between the behavior of microparticles (atoms, molecules) in a medium with traps (in materials science) and the behavior of lymphocytes in living tissue containing tumor cells (in oncology). It is established that the tools of the qualitative theory of differential equations (analogous to those applicable to oncology) can be effective in analysis of transport and transformations in materials science. __________ Translated from Poroshkovaya Metallurgiya, Nos. 1–2(447), pp. 92–98, January–February, 2006.     

Comparative analysis of the cya locus in enterobacteria and related gram-negative facultative anaerobes

Cadmium influx rate in mammal kidney cells (MDCK) is analyzed using an original method based on fura-2 titration. The method relies on the high affinity of the fluorophore for the metal. It follows that the excitation spectrum shift of fura-2 can be linearly correlated to the influx rate of cadmium. Fluorescence digital imaging microscopy allows the study at single cell and intra-cellular organite levels. Results show that the cadmium uptake seems to be carrier dependent. Metal fluxes are potential independent, with a temperature effect caracterized by a Q10 of 2.3 +/- 0.2. No effect of verapamil is noticed; however, cadmium transport is inhibited by external calcium. Apparent dissociation constant for the cadmium uptake is estimated at 4.5 10(-5) M at 20 degrees C. An additional passive transmembrane diffusion process is also evidenced.     

Electrophoretic analysis of polypeptides and glycopeptides of erythrocyte membrane sampled from rats simulating mild insulin dependent diabetes mellitus

In order to understand the molecular mechanism of reduced life span of diabetic erythrocyte, polypeptides and glycopeptides were analyzed by disc gel preparative sodium dodecyl sulphate polyacrylamide gel electrophoresis. An additional glycopeptide (244.5 kDa) and two additional polypeptides (39.81 and 144.5 kDa) were observed on glycopeptide and polypeptide gel profiles of mild insulin dependent diabetes mellitus (mIDDM) sample as compared to control. On the basis of molecular weight, their position on gel profile and their widely accepted nomenclature they were termed as glycosylated-ankyrin, membrane accreted glyceraldehyde-3-phosphate dehydrogenease (G 3-PD) and stress induced band 2.3 peptide. Earlier we have reported an increase in heterogeneity associated with increase in the population of aged fragile cells having altered membrane bound cation dependent ATPases, cytosolic dehydrogenase and hexokinase activities of mIDDM simulating rat erythrocyte sample. Significance of above observation in view of our earlier observation is discussed to explain the molecular mechanism of reduced life span of diabetic erythrocytes.     

Cloning and analysis of a cDNA encoding the BALB/c murine erythrocyte band 7 integral membrane protein

Phospholipid metabolism abnormalities have been suggested by a number of postmortem brain and red blood cell studies in schizophrenia. 31P magnetic resonance spectroscopy enables the examination of phospholipid metabolism in living patients. These in vivo studies have demonstrated that schizophrenic patients have lower prefrontal levels of phosphomonoesters and higher levels of phosphodiesters compared to matched controls. Patients with psychotic depression also seem to show lower levels of phosphomonoesters compared to controls. This suggests that membrane phospholipid differences may not be specific to schizophrenia. Preliminary 31P magnetic resonance spectroscopy studies at high field strength on postmortem temporal lobe samples show no differences between treated schizophrenic patients and controls for phosphoethanolamine and phosphocholine which are the main constituents of the phosphomonoester peak. Further studies are underway in the prefrontal region. While 31P magnetic resonance spectroscopy studies have demonstrated membrane phospholipid abnormalities in schizophrenia, it is not clear whether these findings are specific to schizophrenia or part of a generalized membrane phospholipid abnormality.     

The effects of pentylenetetrazol on molluscan neurons. II. Voltage clamp studies

The effects of pentylenetetrazol (PTZ) upon the steady and transient outward ionic currents during PTZ-induced prolonged depolarizations were investigated using voltage clamp techniques. PTZ causes a 5-35% reduction in gL and a 40-60% reduction in steady-state gK. There is also a marked reduction in the activation of gA of Connor and Stevens6 at all clamp potentials; a shortening of the time constant for the inactivation of gA; and a 10-15 mV shift in the depolarizing direction of the curve relating the steady-state inactivation of gA to membrane potential. The equilibrium potentials for both gA and gK are depolarized by 20 mV in PTZ solution. Equation and voltage clamp data for normal repetitive firing were integrated with the normal and PTZ-alered data. Solution to these equations demonstrated: (1) normal repetitive firing in response to a constant current stimulus; and (2) PTZ-altered repetitive firing that was in the direction of, and for the most part, similar to the observed behavior.     

Comparative pharmacokinetic and cytotoxic analysis of three different formulations of mitoxantrone in mice

Two liposomal formulations of mitoxantrone (MTO) were compared with the aqueous solution (free MTO) in terms of their pharmacokinetic behaviour in ICR mice and cytotoxic activity in a nude mouse xenograft model. The three different formulations of MTO [free MTO, phosphatidic acid (PA)-MTO liposomes, pH-MTO liposomes] were administered intravenously (three mice per formulation and time point) at a dose of 4.7 micromol kg(-1) for free MTO, 6.1 micromol kg(-1) for PA-MTO and 4.5 micromol kg(-1) for pH-MTO. The concentrations of MTO were determined using high-performance liquid chromatography (HPLC) in blood, liver, heart, spleen and kidneys of the mice. Additionally, the toxicity and anti-tumour activity of MTO was evaluated in a xenograft model using a human LXFL 529/6 large-cell lung carcinoma. The dose administered was 90% of the maximum tolerated dose (MTD) of the corresponding formulation (8.1 micromol kg(-1) for free MTO, 12.1 micromol kg(-1) for PA-MTO and pH-MTO). The pharmacokinetic behaviour of PA-MTO in blood was faster than that of free MTO, but the cytotoxic effect was improved. In contrast, pH-MTO showed a tenfold increased area under the curve (AUC) in blood compared with free MTO, without improvement of the cytotoxic effect. This discrepancy between the pharmacokinetic and cytotoxic results could be explained by the fact that MTO in pH-MTO liposomes remains mainly in the vascular space, whereas MTO in PA-MTO liposomes is rapidly distributed into deep compartments, even more so than free MTO.     

Comparative sequence analysis of ribonucleases HII, III, II PH and D

Escherichia coli ribonucleases (RNases) HII, III, II, PH and D have been used to characterise new and known viral, bacterial, archaeal and eucaryotic sequences similar to these endo- (HII and III) and exoribonucleases (II, PH and D). Statistical models, hidden Markov models (HMMs), were created for the RNase HII, III, II and PH and D families as well as a double-stranded RNA binding domain present in RNase III. Results suggest that the RNase D family, which includes Werner syndrome protein and the 100 kDa antigenic component of the human polymyositis scleroderma (PMSCL) autoantigen, is a 3'-->5' exoribonuclease structurally and functionally related to the 3'-->5' exodeoxyribonuclease domain of DNA polymerases. Polynucleotide phosphorylases and the RNase PH family, which includes the 75 kDa PMSCL autoantigen, possess a common domain suggesting similar structures and mechanisms of action for these 3'-->5' phosphorolytic enzymes. Examination of HMM-generated multiple sequences alignments for each family suggest amino acids that may be important for their structure, substrate binding and/or catalysis.