Comparison of early embryonic and differentiating cell surfaces. Interaction of lectins with plasma membrane components

1. Cells of the unincubated as well as those of primitive streak chick blastoderm, which are preparing for or are involved in morphogenetic movements, are agglutinated by wheat germ agglutinin, Ricinus communis agglutinin and concanavalin A, but not by fucose-binding protein. 2. Agglutination of these cells with soybean agglutinin occurs only after neuraminidase treatment, while that induced by concanavalin A, wheat germ and Ricinus communis agglutinins is not affected. 3. Trypsin treatment of blastoderm cells had no effect on lectin-mediated agglutination. 4. In contrast, cells derived from 10-and 12-day differentiating chick liver were agglutinated by wheat germ agglutinin only after trypsinization. 5. Mechanically dissociated embryonic liver cells, which are not agglutinated, bind more 3H-labelled wheat germ agglutinin per cell than trypsinized cells, suggesting that during differentiation there may be a spatial reorganization of wheat germ agglutinin receptors within the plasma membrane. 6. Membranes isolated from the above cell types were examined by analytical polyacrylamide gel isoelectric focusing and, in combination with affinity chromatography using wheat germ agglutinin conjugated to agarose, membrane material in the differentiating liver membrane, which binds to this lectin, was identified.     

Interaction of glucose and metformin with isolated red cell membrane

Isolated human erythrocyte membranes (red blood cell (RBC) ghosts) were incubated with glucose at 5, 10, 20 and 100 mmol/l concentrations, with insulin (0.01 to 200 mU/l) and metformin (CAS 657-24-9) 0.5 up to 50.0 mumol/l). Binding studies with 14C-glucose and subsequent gel electrophoresis revealed 60% of the radioactivity around ban 4.2-4.5 at 5 mmol/l, whereas a random distribution of radioactivity over all protein bands of the RBC membrane was found at 20 mmol/l concentration after incubation for 30 min or 48 h. Metformin does not bind covalently to RBC membranes, however, after photochemical linkage of 14C-metformin via the aminoreactive linker azidophenylglyoxal the highest radioactivity (21%) was counted in the range of band 4.2-4.5. In parallel with an increase of order parameters of 5-doxyl-stearic acid the thiol status of the membranes decreases as determined by monobromobimane fluorescence. 20 and 100 mmol/l concentrations of glucose decrease the reactivity of membrane thiols towards bromobimane significantly to 73 and 62% of the controls. Concomitantly, membrane fluidity at polar sites is diminished as measured by order parameters of spin label 5-doxyl stearic acid. In RBC membranes pretreated with 20 mmol/l glucose the decreased fluorescence is significantly raised again by insulin and metformin. This effect is even more pronounced, if insulin and metformin are incubated together. Reaction of membrane thiols with a maleimido spin label detects modification in the ratio of mobile and immobilized spin label populations in the electron paramagnetic resonance signal under the above conditions, indicative of conformational changes of membrane proteins.     

Interaction of influenza virus haemagglutinin with sphingolipid-cholesterol membrane domains via its transmembrane domain

Sphingolipid-cholesterol rafts are microdomains in biological membranes with liquid-ordered phase properties which are implicated in membrane traffic and signalling events. We have used influenza virus haemagglutinin (HA) as a model protein to analyse the interaction of transmembrane proteins with these microdomains. Here we demonstrate that raft association is an intrinsic property encoded in the protein. Mutant HA molecules with foreign transmembrane domain (TMD) sequences lose their ability to associate with the lipid microdomains, and mutations in the HA TMD reveal a requirement for hydrophobic residues in contact with the exoplasmic leaflet of the membrane. We also provide experimental evidence that cholesterol is critically required for association of proteins with lipid rafts. Our data suggest that the binding to specific membrane domains can be encoded in transmembrane proteins and that this information will be used for polarized sorting and signal transduction processes.     

Interaction of lipophilic ions with the plasma membrane of mammalian cells studies by electrorotation

The electrical properties of biological and artificial membranes were studied in the presence of a number of negatively charged tungsten carbonyl complexes, such as [W(CO)5(CN)]- , [W(CO)5(NCS)]-, [W2(CO)10(CN)]-, and [W(CO)5(SCH2C6H5)]-, using the single-cell electrorotation and the charge-pulse relaxation techniques. Most of the negatively charged tungsten complexes were able to introduce mobile charges into the membranes, as judged from electrorotation spectra and relaxation experiments. This means that the tungsten derivatives act as lipophilic anions. They greatly contributed to the polarizability of the membranes and led to a marked dielectric dispersion (frequency dependence of the membrane capacitance and conductance). The increment and characteristic frequency of the dispersion reflect the structure, environment, and mobility of the charged probe molecule in electrorotation experiments with biological membranes. The partition coefficients and the translocation rate constants derived from the electrorotation spectra of cells agreed well with the corresponding data obtained from charge-pulse experiments on artificial lipid bilayers.     

Interaction of lipids with the plasma membrane of sperm cells. II. Evidence of a membrane thermotropic transition

Cholesterol and phosphatidylcholine uptake from dipalmitoyl-phosphatidylcholine liposomes by rabbit spermatozoa showed a complex dependence on temperature in these experiments. At 5 degrees and 20 degrees C, the rate of lipid uptake correlated with temperature. However, from 20 degrees to 37 degrees C uptake did not evidently increased. The results in interpreted as evidence of a thermotropic transition in the sperm plasma membrane. Data are presented showing incorporation of these lipids, especially that of cholesterol, into sperm plasma membrane.     

Interaction of herpes simplex virus glycoprotein gC with mammalian cell surface molecules

The entry of herpes simplex virus (HSV) into mammalian cells is a multistep process beginning with an attachment step involving glycoproteins gC and gB. A second step requires the interaction of glycoprotein gD with a cell surface molecule. We explored the interaction between gC and the cell surface by using purified proteins in the absence of detergent. Truncated forms of gC and gD, gC1(457t), gC2(426t), and gD1(306t), lacking the transmembrane and carboxyl regions were expressed in the baculovirus system. We studied the ability of these proteins to bind to mammalian cells, to bind to immobilized heparin, to block HSV type 1 (HSV-1) attachment to cells, and to inhibit plaque formation by HSV-1. Each of these gC proteins bound to conformation-dependent monoclonal antibodies and to human complement component C3b, indicating that they maintained the same conformation of gC proteins expressed in mammalian cells. Biotinylated gC1(457t) and gC2(426t) each bind to several cell lines. Binding was inhibited by an excess of unlabeled gC but not by gD, indicating specificity. The attachment of gC to cells involves primarily heparan sulfate proteoglycans, since heparitinase treatment of cells reduced gC binding by 50% but had no effect on gD binding. Moreover, binding of gC to two heparan sulfate-deficient L-cell lines, gro2C and sog9, both of which are mostly resistant to HSV infection, was markedly reduced. Purified gD1 (306t), however, bound equally well to the two mutant cell lines. In contrast, saturating amounts of gC1(457t) interfered with HSV-1 attachment to cells but failed to block plaque formation, suggesting a role for gC in attachment but not penetration. A mutant form of gC lacking residues 33 to 123, gC1(delta 33-123t), expressed in the baculovirus system, bound significantly less well to cells than did gC1(457t) and competed poorly with biotinylated gC1(457t) for binding. These results suggest that residues 33 to 123 are important for gC attachment to cells. In contrast, both the mutant and wild-type forms of gC bound to immobilized heparin, indicating that binding of these proteins to the cell surface involves more than a simple interaction with heparin. To determine that the contribution of the N-terminal region of gC is important for HSV attachment, we compared several properties of a mutant HSV-1 which contains gC lacking amino acids 33 to 123 to those of its parental virus, which contains full-length gC. The mutant bound less well to cells than the parental virus but exhibited normal growth properties.(ABSTRACT TRUNCATED AT 400 WORDS)     

Biochemical evidence that Semliki Forest virus obtains its envelope from the plasma membrane of the host cell

The data from chemical studies and electron microscopy suggest that Semliki Forest virus obtains its envelope by budding into the medium from the plasma membrane of the host cell. Biochemical evidence for this phenomenon, however, has not been published. Therefore, we undertook a series of pulse-chase studies so that we might quantitatively evaluate the importance of the budding mechanism in the morphogenesis of Semliki Forest virus. Baby hamster kidney cells (clone 13) were grown in culture and infected with Semliki Forest virus. The cells were exposed to [4,5-3H] leucine for 20 min and the subsequent incorporation of the label into virus proteins associated with cytoplasmic membranes and extracellular virus was determined. Initial experiments were conducted with microsomes and a precursor-product relationship was demonstrated between viral proteins in the microsomes and in extracellular virus. Further studies were performed with endoplasmic reticulum and plasma membrane preparations. Maximal incorporation of [3H] leucine was observed in the viral proteins located in the endoplasmic reticulum at the end of a 20-min pulse period; greater than 50% of this activity had disappeared within 2 h. The plasma membrane fraction contained no radioactivity at the end of the pulse period; subsequently, maximal labeling of the viral proteins in the plasma membrane occurred 4 h into the chase period and these labeled proteins had disappeared from this membrane 11 h after the pulse. At this time maximal incorporation of the labeled proteins into extracellular virus was observed. These data are consistent with a precursor-product relationship between the viral proteins in the endoplasmic reticulum which migrate to the plasma membrane and are subsequently incorporated into extracellular virus. All the radioactivity in the extracellular virus appears to have been derived from viral proteins associated with the plasma membrane of the cell. Therefore, mechanisms for the morphogenesis of Semliki Forest virus (in baby hamster kidney cells), other than budding from the plasma membrane, are unlikely to be of quantitative importance.     

Interaction of transresveratrol with plasma lipoproteins

OBJECTIVE: To determine electroencephalographic (EEG) changes occurring during syncope induced by headup tilt table testing. DESIGN: Prospective observational study. SETTING: Calgary General Hospital Syncope Clinic, Calgary, Alberta. PATIENTS: Eighteen patients with a history of recurrent syncope who developed syncope while undergoing diagnostic isoproterenol tilt table testing. INTERVENTIONS: Continuous EEGs were recorded in 18 sequentially consenting patients while they underwent diagnostic headup tilt table testing. MAIN RESULTS: Patients developed presyncope after 2.6 +/- 2.4 mins and syncope after 3.7 +/- 2.5 minutes. Systolic blood pressure dropped from 117 +/- 17 mmHg to 65 +/- 9 mmHg, and heart rate dropped from 124 +/- 26 beats/min to 65 +/- 27 beats/min. Fourteen patients developed presyncope, while five developed syncope without appreciable presyncope. Abnormal EEGs were recorded in 13 of 14 patients during presyncope and in 18 of 18 patients during syncope. No patients developed EEG abnormalities before the onset of presyncope, and the proportion of patients with EEG abnormalities gradually increased throughout presyncope. During presyncope, theta and delta wave slowing, and background suppression were noted in eight of 14, nine of 14 and one of 14 patients, respectively. During syncope, theta and delta wave slowing, and background suppression were noted in nine of 18, 11 of 18 and six of 18 patients, respectively (not significant versus presyncope). There were strikingly abrupt changes in the EEG rhythm within 15 s of the transition to syncope in 14 of 18 patients. Six patients developed new theta wave slowing, 11 developed new delta wave slowing, and seven developed background suppression. No epileptiform activity was recorded. CONCLUSIONS: Both presyncope and syncope induced by tilt testing are associated with EEG abnormalities, and no single EEG pattern is pathognomonic of either. The transition from presyncope to syncope is marked by abrupt EEG changes.     

Specific binding of 3n-melatonin with plasma cell membrane in rats thyroid gland

The present study compared the Kato-Katz thick smear and formol ether sedimentation techniques in the diagnosis of Schistosoma mansoni infections. A stool specimen was collected from 915 individuals representing a high prevalence community (63.3%) and from 471 individuals representing a relatively low prevalence village (40%). The overall sensitivity of a single Kato-Katz smear was 70.8%, and it increased with each additional slide to reach 91.7% on examining four smears. However, the sensitivity was 83.3% when using the formol ether sedimentation technique. In terms of quantitative analysis, the geometric mean egg count was 94 eggs per gram (epg) of stool by two Kato-Katz smears, and 43 epg by the sedimentation technique. This means that more than 50% of eggs were missed when using the sedimentation technique, a fact that should be taken into consideration when relating infection level with morbidity.     

Stimulation of tumor-specific immunity using tumor cell plasma membrane antigen

An improved methodology is described for the separation of yolk IgG into subpopulations using immobilized metal ion (Fe3+) affinity chromatography. The yolk IgG was first extracted using a prechilled, pre-acidified method. After extraction, the yolk IgG was then fractionated using an Fe3+ column. Using an ascending pH gradient, four IgG containing peaks were well resolved based upon the elution pH, specific activity and the relative avidity index.     

Potency assay of diphtheria antitoxin in Vero cell microcultures

A slowly rising cortical potential shift with negative polarity following the imperative stimulus of a forewarned reaction time task, the 'post-imperative negative variation' (PINV), is regularly observed in schizophrenic patients but not in controls. The topography of the PINV suggests that it may originate in frontal cortical regions. We used a task designed to test two putative prefrontal cortical functions: working memory and processing of ambiguity. Nineteen patients with a chronic schizophrenic disorder and 19 control subjects matched for age, sex, and education participated in two experimental sessions. The EEG was recorded from frontal, central, temporal, and parietal leads over both hemispheres using a DC amplifier. PINV amplitudes were generally larger in patients than in controls. If the result of comparing physical features of the two successively presented stimuli (warning and imperative stimulus) was ambiguous rather than clear, an augmentation of the PINV amplitudes was seen in both groups. If this comparison required high rather than low involvement of working memory functions, PINV amplitudes were augmented in schizophrenic patients only. Scalp distribution of the PINV indicated a left-hemisphere fronto-central PINV maximum in patients, and a right-hemisphere predominance in controls, which was larger following ambiguous stimulus comparisons. These results suggest that ambiguity during the comparison of physical features of successively presented stimuli may be a general factor of the PINV in schizophrenic patients and healthy controls. Augmented involvement of working memory functions, presumably subserved by the prefrontal cortex, specifically affected the fronto-centrally predominant PINV in schizophrenic patients. This result is compatible with the hypothesis of prefrontal cortical dysfunctions in schizophrenia.     

Interaction of aldolase with pigeon erythrocyte plasma membranes]

Osmotically hemolysed pigeon erythrocytes retain a considerable part of the total cell content of aldolase activity. After washing off the ghosts from hemoglobin and removing the nuclei, a considerable portion of aldolase activity is found in the supernatant. The retained part of aldolase is rather firmly bound to plasma membranes (PM), as evidenced by the fact, that double washing with a mixture of 0.3 M sucrose, 0.01 M tris-HCl (pH 7.4) and 0.004 M MgCL2, or with 0.15 M NaCl or H2O does not appreciably decrease the aldolase activity of PM. Only washing of PM with 0.5 M NaCl results in appreciable decrease of aldolase retention by PM. The binding of aldolase proved to be temperature sensitive: after heating the binding of aldolase to PM specifically decreased. These data suggest that the interaction of the enzyme with PM of pigeon erythrocytes occurs in the intact cell and may be of physiological significance.     

Characterization of defective viral RNA produced during persistent infection of Vero cells with Murray Valley encephalitis virus

Defective interfering viral particles are readily produced in cell culture after a high multiplicity of infection with many animal RNA viruses. Due to defects that they carry in their genomes, their life cycle needs to be complemented by the helper functions provided by a parental virus which makes them both dependent on and competitive with the parental virus. In many instances, this may cause the abrogation of a lytic cycle of the parental virus, leading to a persistent infection. In this paper, we describe for the first time the presence of truncated or defective interfering viral RNAs produced in Vero cells persistently infected with the flavivirus Murray Valley encephalitis virus. While these RNAs have not been detected in acutely infected Vero cells, their appearance coincided with the establishment of persistent infection. We also show for the first time that the defective viral RNAs replicate well in both cell culture and cell-free virus replication systems, indicating that they may interfere with the replication of parental virus at the level of viral RNA synthesis. Significantly, structural analyses of these RNA species including nucleotide sequencing have revealed that they carry similar nucleotide deletions encompassing the genes coding for the prM and E proteins and various gene segments coding for the N terminus of the NS1 protein. These deletions are in frame, allowing the synthesis of truncated NS1 proteins to occur in persistently infected cells. This may have further implications for the interference with the parental virus at the level of viral RNA synthesis in addition to a major one at the level of virion assembly and release.     

Interaction of artificial carbon pyroceram mitral valve with blood plasma

The nanocrystalline material of an artificial carbon pyroceram mitral valve obtained by sintering of 15 wt.% B₄C with crystals <10 nm that are uniformly distributed in 85 wt.% carbon with particles ～10 nm has exceptionally high chemical stability in blood plasma. The electrochemical interaction resulting from contact with a possible microadditive (for example, iron) on the valve surface is experimentally modeled by polarization induced by an external current source specially to create extreme corrosion conditions. The interaction kinetics is studied at 37 °C using anodic polarization curves. Curcumin is used as an analytical reagent for spectrophotometry of boron traces in a solution. Emissive spectroscopy is used to determine iron traces in the spume-like film formed after polarization. It is established that a chemisorbed oxygen film forms when microgalvanic elements appear at 0.4 V and stable passivation of the valve surface is observed at ～1.0 V since a low-conductive nanostructured carbon film forms. It is shown that this film results from the discharge of α-amino acids on the valve surface (amino acid residues of complex peptide chains of plasma proteins) containing heterocyclic rings. The sessile drop method shows that the valve is promptly wetted by blood plasma (wetting angle is 50 °), this also promotes the formation of a stable protective film on its surface.     

Interaction of Theiler's virus with intermediate filaments of infected cells

Theiler's murine encephalomyelitis virus is a neurotropic murine picornavirus which replicates permissively and causes a cytopathic effect in the BHK-21 cell line. We examined the interactions between the GDVII and DA strains of Theiler's virus and BHK-21 host cell proteins in a virus overlay assay. We observed binding of the virions to two proteins of approximately 60 kDa. These proteins were microsequenced and identified as desmin and vimentin, two main components of the intermediate filament network. The association between desmin or vimentin and virions was demonstrated by immunoprecipitation. Anti-desmin and anti-vimentin monoclonal antibodies precipitated GDVII or DA virions from extracts of infected BHK-21 cells. The intracellular distributions of virions and of the desmin and vimentin intermediate filaments of BHK-21 cells were investigated by two-color immunofluorescence confocal microscopy. Following infection, the intermediate filament network was rearranged into a shell-like structure which surrounded a viral inclusion. Finally, close contact between GDVII virus particles and 10-nm intermediate filaments was observed by electron microscopy.