Human serum-sensitive Trypanosoma brucei rhodesiense: a comparison with serologically identical human serum-resistant clones.

Trypanosoma brucei rhodesiense clones, which are susceptible to lysis by normal human serum, were isolated from 3 different human serum-resistant clones originally derived from strain ETat 1.10. Serologically, these pairs of serum-sensitive and serum-resistant clones displayed the same variant surface glycoprotein (VSG) on their surface. Acquisition of human serum sensitivity correlated with susceptibility to lysis by human high density lipoprotein, a trypanocidal factor in normal human serum. Analysis of these paired populations by two-dimensional gel electrophoresis of whole trypanosomes and various subcellular fractions failed to reveal any differences in mobility of VSG and other proteins. Northern blot analysis of mRNAs from serum-sensitive and serum-resistant clones showed no differences when probed with a previously described resistance-specific probe. In addition, the ethanolamine membrane transport system and the overall membrane lipid fluidity did not reveal any detectable biochemical or biophysical differences in membrane properties. If resistance to lysis is indeed mediated by membrane changes at the enzymatic or structural level, the data presented suggest that the gene product(s) responsible for this change in human serum sensitivity may be present in very small quantities.

Interferon-alpha modulates the plasma membrane-cytoskeletal complex of human lymphoblastoid cells sensitive to the antiproliferative action of interferon-alpha.

Interferon (IFN)-induced structural changes in the plasma membrane-cytoskeletal complex were investigated in human lymphoblastoid cell lines that differed in sensitivity to the antiproliferative action of IFN-alpha. These cell lines have structurally indistinguishable high-affinity IFN-alpha receptors. By using electron spin resonance techniques, we determined the effect of IFN-alpha on fluidity of the plasma membrane lipid bilayer. By using the ability of cells to redistribute fluorescent anti-immunoglobulin (Ig) into caps at the cell surface, we investigated the effect of IFN-alpha on the mobility of the cell surface. IFN-alpha decreases the fluidity of the plasma membrane lipid bilayer of lymphoblastoid cells sensitive to the antiproliferative action of IFN-alpha. Similarly, IFN-alpha inhibits the anti-Ig-induced redistribution of surface immunoglobulins (capping) of IFN-sensitive cells. These effects of IFN-alpha occur within the first few hours after IFN-alpha addition to cells. However, IFN-alpha had no effect on membrane fluidity or anti-Ig-induced capping of lymphoblastoid cells resistant to the antiproliferative action of IFN-alpha.

Trypanosome variant surface glycoprotein transfer to target membranes: a model for the pathogenesis of trypanosomiasis.

The variant surface glycoprotein (VSG) of trypanosomes is attached to the cell surface by means of a phosphatidylinositol-containing glycolipid membrane anchor. The studies presented in this paper support the hypothesis that the transfer of VSG from trypanosomes to erythrocytes could lead to one of the pathological features associated with trypanosome infection--i.e., anemia. Migration of trypanosome VSG from live trypanosomes to target cells (sheep erythrocytes) could be shown by preincubating erythrocytes with trypanosomes and subsequently testing the washed erythrocytes for insertion of VSG by their susceptibility to lysis by complement in the presence of an anti-VSG antibody. Complement-mediated lysis was found to depend on the strain-specific anti-VSG antibody used. Extent of erythrocyte lysis increased with time of cell exposure to trypanosomes and with trypanosome concentration. No erythrocyte lysis was observed when trypanosomes were preincubated with anti-VSG antibody before adding erythrocytes. Purified membrane-form VSG (which retains the glycolipid anchor), but not soluble VSG (which no longer has the terminal diacylglycerol moiety), could sensitize erythrocytes to anti-VSG antibody-mediated complement lysis. The intermembrane transfer of VSG from trypanosomes to cells of the infected host could provide a molecular mechanism for the pathogenesis of trypanosomiasis.

Primary structural repeats in human and in murine gamma-interferon.

The Sellers TT algorithm has been used to conduct a detailed pattern search in the amino acid sequence of human and murine gamma-interferon. Each of these proteins contains two large repeats at the level of the primary amino acid sequence that divide each of these molecules into an aminoterminal and a related carboxyterminal segment.

Significance of similarities in patterns: an application to beta interferon-related DNA on human chromosome 2.

The nucleotide sequence of a 14-kilobase (kb) region of the human beta interferon (IFN-beta)-related DNA locus on chromosome 2 (genomic DNA clone lambda B3) was determined and compared to that of the IFN-beta 1 gene by using the Sellers TT algorithm. This algorithm aligns segments of one sequence with similar segments in a second sequence. A strategy was developed for assessing the significance of similarities between DNA sequences based on a scheme that recognizes patterns or runs of identities within an alignment. The pattern score (II) thus obtained is an entropy-like measure. Numerically it is a reflection of the length of the second longest run of identity in an alignment plus a correction factor due to the other shorter identity runs in the alignment. When the IFN-beta 1 gene is compared to a random nucleotide sequence, the distribution of II scores in such comparisons fits a Gaussian function. This strategy has been used to identify seven segments along one strand of lambda B3 DNA that are related to segments in IFN-beta 1; these seven alignments have II scores greater than or equal to 3 standard deviations above the mean score obtained in comparisons between IFN-beta 1 and random nucleotide sequences. One of these alignments (section 7) has a II score 8.02 standard deviations above this mean score. The likelihood of finding an alignment statement as good as that in section 7 in a random sequence the length of the human genome is approximately 10(-7). Furthermore, the lambda B3 DNA sequence in section 7 selects the human IFN-beta 1 gene as the most significant alignment in computer searches of mammalian nucleotide sequence data bases.

Interferon stimulates cholesterol and phosphatidylcholine synthesis but inhibits cholesterol ester synthesis in HeLa-S3 cells.

Treatment of human HeLa-S3 cells (an epidermoid carcinoma line) with human beta-interferon (640 units/ml) selectively alters lipid metabolism by increasing cholesterol synthesis per mg of cell protein as measured by 1-hr pulse-labeling of cells with [3H]acetate. Cholesterol synthesis in interferon-treated cells is increased approximately equal to 60% at 24 hr after the beginning of treatment and approximately equal to 450% at 48 hr. Continuous labeling of interferon-treated cells with [14C]acetate shows increased accumulation of label in cholesterol when normalized per mg of cell protein, as well as an increase in the specific activity of cholesterol in the treated cells. In contrast, interferon treatment decreases the accumulation of [14C]acetate into cholesterol esters. The [14C]acetate labeling of sphingomyelin, phosphatidylethanolamine, and triglycerides shows no change compared to untreated controls. The labeling of phosphatidylcholine was moderately increased in treated cells. The interferon-induced changes in lipid metabolism are a part of a coordinated response of cells to interferon treatment, characterized by reduced cell proliferation and cell motility and an increase in cell size and mass. The increased cholesterol synthesis is consistent with a model in which beta-interferon treatment of HeLa cells inhibits the endocytosis of cholesterol-containing low density lipoprotein, which results in an increase in cholesterol synthesis.

Occurrence of cell division is not exponentially distributed: differences in the generation times of sister cells can be derived from the theory of survival of populations.

The Eyring-Stover survival theory has been applied to the kinetics of the distribution of intermitotic intervals of mammalian cells and by inference to the transition from the G1 phase into DNA synthesis (S phase). The theory faithfully fits experimental data acquired by time-lapse cinemicrography of cloned HeLa cells in tissue culture and also suggests the existence of a labile initiator substance which mediates the G1-S phase transition. This theory provides an alternative to the transition probability model proposed by Smith and Martin [Smith J. A. & Martin, L. (1973) Proc. Natl. Acad. Sci. USA 70, 1263-1267; see also Shields, R. & Smith, J. A. (1977) J. Cell Physiol. 91, 345-355 and Brooks, R. F., Bennett, D. C. & Smith, J. A. (1980) Cell 19, 493-504].

Structural changes in BHK cell plasma membrane caused by the binding of vesicular stomatitis virus.

Spin label electron spin resonance techniques using a nitroxide derivative of stearic acid were used to detect changes in plasma membrane structure caused by the binding of vesicular stomatitis virus (VSV) to cell plasma membranes of intact BHK-21 cells. The results indicate that binding of VSV to cell surface receptors causes an increase in the observed rigidity of the plasma membrane lipid bilayer. This change in membrane structure, which appears to be caused by the cross-linking of receptors in the plane of the plasma membrane, could be prevented by treating the cells with colchicine before addition of virus and could be reversed by treating the cells with colchicine after addition of virus. Cells treated with a monovalent, water-soluble derivative of VSV G-protein (Gs) did not show an increase in plasma membrane bilayer rigidity. However, addition of anti-VSV G-protein immunoglobulin G to cells pretreated with G8 caused an increase in plasma membrane bilayer rigidity. This increased rigidity could also be reversed by the addition of colchicine. Fluorescence microscopy was used to determine the distribution of fluorescein-labeled VSV particles on the cell surface after addition of virus. Approximately 30 min after addition of virus, discrete areas on the cell surface showed fluorescent staining, which coalesced to apical regions of the cell after approximately 40 min.

Beta-interferon-induced time-dependent changes in the plasma membrane lipid bilayer of cultured cells.

The time-dependent interferon-induced structural changes in the plasma membrane lipid bilayer have been investigated in cultured cells using spin label electron spin resonance techniques. Treatment of human HeLa-S3 cells in suspension culture with human beta 1 interferon (640 u/ml) for as short a time as 30 min causes an increase in the rigidity of the plasma membrane lipid bilayer (Landsberger, Pfeffer and Tamm, in preparation). The plasma membrane rigidity of interferon-treated cells returns to the level of control cells within 3 to 5 hr, but by 24 hr after beginning of treatment, the rigidity of the plasma membrane is increased again and remains so for at least 2 days. Mouse beta interferon causes both an early (Landsberger, Pfeffer and Tamm, in preparation) and a late increase in membrane rigidity in homologous mouse L-929 cells, but not in heterologous HeLa-S3 cells. Thus, the interferon-induced perturbation of membrane structure is species specific. The late increase in membrane rigidity may have a different underlying mechanism from that of the early increase. While the early and transient change probably is related to signal generation and transmission, the later and persistent change may be an aspect of the phenotype and interferon-treated cells and may reflect changes in the plasma membrane-cytoskeletal complex.

Kinetics of desynchronization and distribution of generation times in synchronized cell populations.

The kinetics of the distribution of generation times in synchronized cells can be analyzed by using Fourier transform analysis. Deviations of the experimental data from the curve of completely asynchronous growth reflect the degree of synchrony at a particular time. Fourier transform analysis of these deviations yields the average generation time as well as information on the distribution of generation times characterizing a synchronized culture. A detailed analysis of synchronized cell cultures does not provide any evidence for the existence of a subcycle or a polymodal distribution in generation times. The data do indicate that cell-cell interaction occurs at cell densities as low as 2.5 X 10(3)/cm2. It is also shown that the Eyring-Stover formalism for the dynamics of survival can correctly describe the distribution of the first round of cell divisions in a synchronized culture.

Effects of filipin on the structure and biological activity of enveloped viruses.

The interaction of the polyene antibiotic filipin with membrane-bound cholesterol in vesicular stomatitis (VS), influenza, and Rauscher leukemia virions was studied. Exposure of virions to filipin resulted in a series of depressions and ridges in the envelope of VS virions, with a periodicity of 15 to 20 nm perpendicular to the long axis of the particle; similar morphological alterations were observed in negatively stained preparations, in thin-sectioned virions, and in protease-treated virions that lack surface glycoproteins. This morphological effect was specific for filipin, since the envelopes of VS virions that had been treated with another polyene antibiotic, amphotericin B, exhibited markedly different morphology. Morphological alterations induced by filipin in influenza and Rauscher leukemia virions differed from those seen in VS virions. The infectivity of filipin-treated VS virions was reduced up to 500-fold, whereas influenza virions were resistant to filipin treatment. Incorporation of filipin into the virions was demonstrated, and no release of either lipids or proteins from virions was detected after filipin treatment. A stoichiometry of approximately 1 mol of bound filipin per mol of cholesterol was found in both intact and protease-treated VS virions. The equilibrium dissociation constant for filipin-cholesterol interaction was approximately 74-fold larger in intact than in protease-treated VS virions. The initial rate of association of filipin with cholesterol in intact virions was slower than that in protease-treated particles. The fluidity of lipids in VS viral membranes, as probed by a stearic acid derivative spin label, was markedly reduced when either intact or protease-treated virions were treated with filipin.

Sendai virus-induced hemolysis: reduction in heterogeneity of erythrocyte lipid bilayer fluidity.

Hemolysis of human or chicken erythrocytes by Sendai virus causes a change in the structure of the erythrocyte membrane lipid bilayer that can be detected by spin label electron spin resonance. In the intact erythrocyte, the phosphatidylcholine derivative spin label exists in a more rigid environment than the corresponding phosphatidylethanolamine label. Virus-induced hemolysis tends to abolish this difference in fluidity, i.e., the region of the phosphatidylcholine spin label becomes more fluid and that of the phosphatidylethanolamine spin label becomes more rigid. Fatty acid derivative spin labels, which may detect some "average" environment, show no change in fluidity. The fluidity change is detected at several different positions in the fatty acyl chain of the phosphatidylcholine spin label. Sendai virions grown in Madin-Darby bovine kidney (MDBK) cells or grown in eggs and harvested early, which lack hemolytic activity, cause no significant change in bilayer structure. Hemolytic activity and the ability to alter erythrocyte bilayer fluidity can be activated in MDBK-grown Sendai virions by trypsin treatment in vitro and in early-harvest egg-grown Sendai virions by freezing and thawing. Erythrocyte ghosts prepared by osmotic hemolysis and resealed by treatment with Mg2+ or elevated ionic strength exhibit a difference in fluidity between phosphatidylcholine and phosphatidylethanolamine spin labels, although less than that observed in whole cells. Incubation of resealed ghosts with Sendai virus abolishes the difference in fluidity. Unsealed ghosts that have been extensively washed show no heterogeneity in membrane bilayer fluidity, and incubation with Sendai virus causes no further fluidity change. Virus-induced hemolysis as measured by hemoglobin release is more sensitive to inhibition by Ca2+ than is the associated fluidity change in the bilayer.