Spatially periodic discrete contact regions in polylysine-induced erythrocyte-yeast adhesion

Cell-cell adhesion occurs when human erythrocytes and yeast cells are suspended together in suprathreshold concentrations of polylysine in saline. The threshold polycation concentration for adhesion depends on cell concentration and decreases with increasing polycation molecular weight. The threshold concentration was similar for erythrocyte-erythrocyte adhesion and for yeast-erythrocyte adhesion. Transmission electron micrographs show that the erythrocytes adhere to yeast as if to engulf the cell. The regions of close contact between the erythrocyte membrane and the yeast cell walls are spatially discrete. The contact separation distance for the asymmetric erythrocyte-yeast adhesion is very similar to that (0.83 micron) observed when polylysine-induced adhesion occurs in the symmetrical erythrocyte-erythrocyte system. The spacing is attributed to the growth of a squeezing wave as an interfacial instability, on the intercellular aqueous layer. Freeze-fracture electron microscopy of cells that were not fixed during preparation for microscopy confirms the discrete nature of contacts between polylysine treated erythrocytes.     

Contact patterns in concanavalin A agglutinated erythrocytes.

Agglutination of human erythrocytes by the lectin concanavalin A is enhanced when the erythrocytes are pretreated with neuraminidase, which removes sialic acids, or with pronase, which degrades both the glycophorins and band 3 protein. In the present work transmission electron microscopy of the enzymatically pretreated erythrocytes shows a regular pattern of interruption of contact between interacting plasma membranes. The lengths characteristic of the pattern were 0.66 and 0.50 microns for pronase- and neuraminidase-pretreated cells, respectively. Agglutination of normal erythrocytes and of neuraminidase-pretreated erythrocytes can be fully reversed by exposure to the competitive inhibitor methyl alpha-D-mannopyranoside. Complete reversal of contact does not occur with pronase-pretreated cells. The comparatively greater tenacity of contact between cells that were treated with pronase before exposure to lectin argues for an involvement of nonspecific interactions in the agglutination process. The results are compared with previously published studies of spatially periodic contact patterns induced by a range of other polymers.     

Spreading of wheat germ agglutinin-induced erythrocyte contact by formation of spatially discrete contacts.

The time dependence of agglutination and cell-cell contact spreading in human erythrocytes exposed to wheat germ agglutinin (WGA) was characterized by light and electron microscopy. Cells (3 x 10(7)/mL) had a threshold lectin concentration in the range of 0.6-2.0 micrograms/mL for initial cell contact. Spreading was essentially completed within 60 and 2 min in undisturbed and gently agitated suspensions, respectively. The cells in large WGA agglutinates retained features of their initial disk form in contrast to the convex outlines of polycation or polyethylene glycol-induced agglutinates. Spreading of contact area was accompanied by development of a pattern of discrete contact regions separated by a distance of the order of 1 micron. Freeze fracture electron microscopy and studies with ferritin-labeled WGA showed no significant aggregation of intramembrane particles or specific lectin receptors under conditions when contact spreading occurred. It is argued that flow stress effects on cells in suspended agglutinates give rise to a situation where opposite membranes, at the leading edge of cell contact, are separated by a thin aqueous layer. When this intercellular water layer exceeds a critical length, it becomes unstable. The layer breaks up by surface wave development to form an array of intracellular water spaces. Formation of the aqueous spaces causes opposite membrane regions to move synchronously toward each other. Lectin molecules crosslink the wave crests to give spatially periodic contact points.     

Comparative effects of trichothecene mycotoxins on bovine platelet function: Acetyl T-2 toxin,a more potent inhibitor than T-2 toxin

The effects of the trichothecene mycotoxins (acetyl T-2 toxin, T-2 toxin, HT-2 toxin, palmityl T-2 toxin, diacetoxyscirpenol (DAS), deoxynivalenol (DON), and T-2 tetraol) on bovine platelet function were examined in homologous plasma stimulated with platelet activating factor (PAF). The mycotoxins inhibited platelet function with the following order of potency: acetyl T-2 toxin > palmityl T-2 toxin = DAS > HT-2 toxin = T-2 toxin. While T-2 tetraol was completely ineffective as an inhibitor, DON exhibited minimal inhibitory activity at concentrations above 10×10^?4M. The stability of the platelet aggregates formed was significantly reduced in all mycotoxin treated platelets compared to that of the untreated PAF controls. It is suggested that the increased sensitivity of PAF stimulated bovine platelets to the more lipophilic mycotoxins may be related to their more efficient partitioning into the platelet membrane compared to the more hydrophilic compounds.     

Depletion flocculation and depletion stabilization of erythrocytes

At dextran (Mw ≈ 500,000) concentrations from 2 to ≈10%, suspensions of normal human erythrocytes flocculate in small convex agglutinates. At dextran concentrations > 10%, the erythrocytes resegregate in a stable monodisperse suspension. At all these dextran concentrations, the erythrocytes are coated with considerable amounts of dextran. It can be argued that at dextran concentrations from 2 to 10%, as well as at dextran concentrations > 10%, there is a thin layer, which is depleted of dextran, between the dextran layer adsorbed onto the erythrocytes and the bulk dextran solution. It can also be shown that there is a repulsive interaction between the two layers of dextran: one adsorbed and one free. When the adsorbed dextran layer is the most concentrated, stability must ensue, and when the dextran in free solution is the most concentrated, flocculation should occur. Below 7% dextran, the concentration of free dextran is higher than the adsorbed concentration; above 10% dextran that situation is reversed. These data correlate well with the depletion flocculation predicted for the lower concentration and the depletion stabilization predicted for the higher dextran concentration.

     

An extensive review on antifungal approaches in the treatment of mucormycosis

During the period of COVID-19, the occurrences of mucormycosis in immunocompromised patients have increased significantly. Mucormycosis (black fungus) is a rare and rapidly progressing fungal infection associated with high mortality and morbidity in India as well as globally. The causative agents for this infection are collectively called mucoromycetes which are the members of the order Mucorales. The diagnosis of the infection needs to be performed as soon as the occurrence of clinical symptoms which differs with types of Mucorales infection. Imaging techniques magnetic resonance imaging or computed tomography scan, culture testing, and microscopy are the approaches for the diagnosis. After the diagnosis of the infection is confirmed, rapid action is needed for the treatment in the form of antifungal therapy or surgery depending upon the severity of the infection. Delaying in treatment declines the chances of survival. In antifungal therapy, there are two approaches first-line therapy (monotherapy) and combination therapy. Amphotericin B ( 1 ) and isavuconazole ( 2 ) are the drugs of choice for first-line therapy in the treatment of mucormycosis. Salvage therapy with posaconazole ( 3 ) and deferasirox ( 4 ) is another approach for patients who are not responsible for any other therapy. Adjunctive therapy is also used in the treatment of mucormycosis along with first-line therapy, which involves hyperbaric oxygen and cytokine therapy. There are some drugs like VT-1161 ( 5 ) and APX001A ( 6 ), Colistin, SCH 42427, and PC1244 that are under clinical trials. Despite all these approaches, none can be 100% successful in giving results. Therefore, new medications with favorable or little side effects are required for the treatment of mucormycosis.     

Divalent cation effects on membrane bending in heated erythrocytes

The thermal fragmentation of human erythrocytes involves either surface wave growth and membrane externalization at the cell rim or membrane internalization at the cell dimple. In symmetrical monovalent electrolytes an increase in membrane internalization at the cell dimple correlates with the decrease in zeta potential arising from surface charge (sialic acid residue) depletion. The influence of divalent cations on thermal fragmentation is examined in this work. The erythrocyte zeta potential decreased when divalent cations replaced some Na⁺ in the cell-suspending phase. The incidence of membrane internalization increased in rank order Ca²⁺>Ba²⁺>Mg²⁺Sr²⁺. Calcium continued to influence the thermal fragmentation of cells highly depleted of sialic acid, suggesting that the ion also interacted with membrane sites other than sialic acid. The divalent cation influence on cell fragmentation was shown to be greater than that due to zeta potential decrease alone. This conclusion was supported by the observation that the divalent cation-induced changes in zeta potential showed much less cation specificity than did the changes induced in the thermal fragmentation pattern. The result implies that the specificity of the divalent cation effects was due to interactions within the erythrocyte shear layer. The possibility that the interaction is with membrane lipids is examined.     

Membrane-membrane interactions: parallel membranes or patterned discrete contacts.

Theoretical and experimental studies of thin liquid films show that, under certain conditions, the film thickness can undergo a sudden transition which gives a stable narrower film or ends in film rupture at spatially periodic points. Theoretical analysis have also indicated that similar transitions might arise in the thin aqueous layer separating interacting membranes. Experiments described here show spatially periodic intermembrane contact points and suggest that spontaneous rapid growth of fluctuations can occur on an intermembrane water layer. Normal and pronase pretreated erythrocytes were exposed to 2% Dextran (450,000 Mr) and the resultant aggregates were examined by light and transmission electron microscopy. Cell electrophoresis measurements were used as an index of pronase modification of the glycocalyx. Erythrocytes exposed to dextran revealed a uniform intercellular separation of parallel membranes. This equilibrium between attractive and repulsive intermembrane forces is consistent with the established Derjaguin, Landau, Verwey, Overbeek (DLVO) model for colloidal particle interaction. In contrast to the above uniform separation a spatial pattern of discrete contact regions was observed in cells coming together in dextran following pronase pretreatment. The lateral contact separation distance was 3.0 microns for mild pronase pretreatment and decreased to 0.85 micron for more extensive pronase pretreatments. The system examined here is seen as a useful experimental model in which to study the principles involved in producing either uniform separation or point contacts between interacting membranes.     

Monoclonal antibodies to rabbit liver cytochrome P-448

Cytochrome P-448 (mol wt 55,000 Daltons) from rabbit liver was purified to a specific content of 16.6 nmol/mg. Mice were immunised with this preparation, their spleens removed and dissociated lymphocytes hybridised with myeloma cells. Four monoclonal antibodies against cytochrome P-448 were raised and partially characterised. All four antibodies interacted with cytochrome P-448 in intact microsomal fractions and selectively immunoadsorbed cytochrome P-448 from solubilised microsomal preparations. One of the antibodies inhibited benzo[a] pyrene hydroxylase activity in a reconstituted system, one had no effect on activity and two increased activity. The possible applications of such antibodies are discussed.