Glutaraldehyde crosslinking of red blood cells by using a hemodialyzer

Summary Human red blood cells (RBC) were crosslinked with glutaraldehyde (GA) by using a hemodialyzer which is used as an artificial kidney. Human RBC, which was in a flow of 2 ml/min, was extensively crosslinked with 50 mM GA solution of 10 ml/min flow rate. The crosslinked RBC showed high stability against osmotic pressure. The oxygen transport activity of the crosslinked RBC was similar to unmodified RBC. This crosslinking method could be used for the development of an efficient reactor which produces a stable and active RBC.     

Comparison of the cryoprotection of red blood cells by 1,2-propanediol and glycerol

Red blood cells are cooled in buffered solutions containing 10, 15, 20, 30, or 35% ($\text{w}\text{w}$) 1,2-propanediol or glycerol. Cell survival is measured after cooling to ?196 °C at rates between 1 and 3500 °C/min, followed by rewarming rapidly, except in a few cases. At low cooling rates, where the injuries are due to solution effects, for the same ($\text{w}\text{w}$) concentrations of 15 or 20% ($\text{w}\text{w}$), 1,2-propanediol protects erythrocytes better than glycerol. Differences are still observed when the two cryoprotectants are compared on a mole-fraction basis. At high cooling rates the survival passes through a minimum and then increases again. For the same concentrations, the minimum occurs at much lower cooling rates with 1,2-propanediol than with glycerol, in agreement with the better glassforming tendency of 1,2-propanediol solutions. These cooling rates almost coincide with those at which the quantity of ice crystallized begins to decrease in the corresponding solutions. Thus, survival seems to be closely related to the glass-forming tendency at the survival minimum, and at higher cooling rates. After the fastest cooling rates, the warming rates necessary to avoid damage on warming are much smaller than those necessary to avoid devitrification. Therefore, in the present experiments the survivals are not related to the stability of the wholly amorphous state. However, injury follows the presumed transition from cubic to hexagonal ice, in erythrocytes as well as in other kinds of cells.     

Induction of giant cells by the synthetic food colorants viz. lemon yellow and orange red

Cytotoxicity and giant cell formation induced by lemon yellow and orange red synthetic food colorants were evaluated in the present study. The aqueous solutions of both the dye solutions were tested for cytotoxicity using Allium cepa assay. Frequency of giant cells were determined after treating the root tips with different concentrations of both food colorant solutions viz., 0.005, 0.01, 0.05, 0.1 % for varying time durations (1/2, 1, 2, 3 h). These colorants may cause giant cell formation primarily by interfering with the normal course of mitosis. Giant cells showing multiple aberrations viz. bridged and binucleate condition, cellular fragmentation, nuclear lesion, double and multiple nuclear lesions, double nuclear peaks and cellular breakage, elongated nucleus, nuclear budding, hyperchromasia, micronucleus, nuclear erosion, pulverized nucleus etc. were induced in root tips treated with both of the colorants. The synthetic food colorant treated cells showed inhibition of cell division and induction of giant cells. A dose dependant decrease in the mitotic index [88.20 % (c^?ve, 3h) to 81.54 % (Lx₄, 3h) and 88.20 % (c^?ve, 3h) to 73.17 % (Ox₄, 3h)] was observed. All mitotic phases show significant induction of giant cells when treated with both food colorants. Interphase stage shows higher percentage of giant cells, whereas in cytokinesis it was negligible. The orange red food colorant is observed to be more toxic because it recorded higher percentage of giant cell induction when compared with lemon yellow [27.93 % (Lx₄, 3h) and 28.07 % (Ox₄, 3h)].     

Comparison of tyrosinase levels in amelanotic and melanotic melanoma cell cultures by a competitive enzyme-linked immunoadsorbent assay and by immunotitration analysis

Melanogenesis in mammalian pigment cells is regulated by changes in the activity of tyrosinase, the rate-limiting enzyme for melanin synthesis. Because recent evidence suggests that this enzyme may exist in pigment cells in both active and inactive stages, a competitive enzyme-linked immunoadsorbent assay (ELISA) was developed to compare tyrosinase levels in amelanotic and melanotic melanoma cell clones. The melanotic cell line used for this study, MEL-11A, had basal tyrosinase levels approximately 40 times that of the amelanotic cell line, AM-7. Both cell lines responded to melanocyte-stimulating hormone by demonstrating large increases in tyrosinase activity. For competitive ELISA analysis of tyrosinase levels in these two clones, microtiter plates were coated with purified tyrosinase, and trypsinized cell extracts were tested for their ability to compete with bound tyrosinase for antibody binding. Although tyrosinase activity in the amelanotic clone was 1/40 that of the melanotic clone, immunoreactive tyrosinase levels in AM-7 cells were found to be approximately one-half that present in the melanotic clone. Additional evidence for the presence of an inactive (or at least, catalytically less active) enzyme in AM-7 cells was obtained from immunotitration analysis of tyrosinase in cell extracts from both cell lines. These results suggest that at least some amelanotic melanoma cells may contain significant levels of catalytically inactive tyrosinase molecules and that the level of pigmentation in mammalian melanocytes may be regulated by a tyrosinase activation process.     

Angiostrongylus cantonensis: preparation of a specific antigen using immunoadsorbent columns.

Antigenic components which reacted specifically with sera of rats infected with Angiostrongylus cantonensis were prepared from adult worm extract by the use of immunoadsorbent columns. Components common to rat serum were removed from a whole worm extract by passing it through a column of cyanogen bromide-activated Sepharose coupled to rabbit antirat immunoglobulin. Consequently, components common to Ascaris suum were similarly removed using Sepharose linked to immunoglobulin to A. suum. Finally, the sample was applied to a column of Sepharose coupled with immunoglobulin from A. cantonensis infected rats and eluted with glycine-hydrochloric acid buffer. Intradermal skin tests using this preparation gave sensitive and specific reactions.     

Congo red absorption and cellulose synthesis by Rhizobiaceae

Congo red uptake by Rhizobium colonies from yeast extract-mannitol-mineral salts-Congo red-agar plates was related with the cellulose content in the cell capsule of the bacteria.     

Fission and refusion of red blood cells using a micropipette technique

In micropipette experiments with small capillaries and moderate high pressure difference (approximately 1000 Pa) cell fragmentation (fission) of human red blood cells without hemolysis was observed by TV-system for a large number of fresh red blood cells of different donors. After separation, the fragment moves away from the residual cell. In seven cases this process was evaluated quantitatively and was shown that the rate of the fragment was constant in time. Two mechanisms for this phenomenon are discussed. In particular cases a spontaneous re-fusion with the residual cell body in the capillary can be observed. In our opinion probably protein-depleted membrane surfaces arise and membrane fusion is possible simply by mechanical contact without additional electric fields and/or fusion agents.     

Continuous determination of protease during the fermentation using the autoanalyzer adm 300

A method for automatic determination of proteolytic activity during fermentation is described. Therefore the autoanalyzer ADM 300 of the PRUEFGERAETEWERK MEDINGEN F.R.G. is connected to a fermenter. After filtration of the biomass, the culture filtrate is diluted and then incubated with casein solution. (0.75%, pH 8.0, 55°C) low moleculare cleavage products are separated from higher molecular ones by dialysis. The absorbence is measured at 600 nm after addition of alkaline copper tartrate and FOLIN solution. By use of different dilution rates, a range of 0–32 units ml^?1 may be measured. The coefficient of variation is 5.5.     

Detection of the absorption of glucose molecules by living cells using atomic force microscopy

A very small electrode (nanobiosensor) was constructed by immobilizing enzyme (glucose oxidase or hexokinase) on the surface of the cantilever of the atomic force microscope in order to detect the absorption of glucose molecules by living cells. If glucose is present, the nanobiosensor deflects, probably due to the reaction heat evolved in the process. Nanobiosensors built with inactivated enzyme or cantilevers without immobilized enzyme were not capable of producing this type of signal (deflection). This technique will be very useful in detecting the passage of specific molecules through a cell wall (or a cell membrane for other types of cells).     

A mathematical model for drug administration by using the phagocytosis of red blood cells

 A mathematical model for the delivery of drug directly to the macrophages by using the phagocytosis of senescent red blood cells is proposed. The model is based on the following assumption: At time t=0 a preassigned red blood cell population n(0, a)=φ(a), a>0, loaded by the drug, is injected in the blood circulation. Among the cells of that population only those with an age a≧ā (ā=120 days) will be phagocytosed by macrophages. Of course, the lifetime of the drug must be higher than ā. Within the red blood cells it cannot be metabolized, neither can it diffuse through their membranes. The emphasis of the paper is on the mathematical properties and on the formulation of the control problem. Received 15 December 1994; received in revised form 20 July 1995     

Comparison of mRNA-display-based selections using synthetic peptide and natural protein libraries

mRNA display is a genotype-phenotype conjugation method that allows the amplification-based, iterative rounds of in vitro selection to be applied to peptides and proteins. Compared to prior protein selection techniques, mRNA display can be used to select functional sequences from both long natural protein and short combinatorial peptide libraries with much higher complexities. To investigate the basic features and problems of using mRNA display in studying conditional protein-protein interactions, we compared the target-binding selections against calmodulin (CaM) using both a natural protein library and a combinatorial peptide library. The selections were efficient in both cases and required only two rounds to isolate numerous Ca2+/CaM-binding natural proteins and synthetic peptides with a wide range of affinities. Many known and novel CaM-binding proteins were identified from the natural human protein library. More than 2000 CaM-binding peptides were selected from the combinatorial peptide library. Unlike sequences from prior CaM-binding selections that correlated poorly with naturally occurring proteins, synthetic peptides homologous to the Ca2+/CaM-binding motifs in natural proteins were isolated. Interestingly, a large number of synthetic peptides that lack the conventional CaM-binding secondary structures bound to CaM tightly and specifically, suggesting the presence of other interaction modes between CaM and its downstream binding targets. Our results indicate that mRNA display is an ideal approach to the identification of Ca2+-dependent protein-protein interactions, which are important in the regulation of numerous signaling pathways.     

Deformation and flow of red blood cells in a synthetic lattice: evidence for an active cytoskeleton.

We introduce the use of microfabrication techniques to construct on a silicon wafer a synthetic capillary bed with 2.5- to 4-micron (mu)-wide channels. Establishment of a fluid pressure gradient allowed us to observe simultaneously using optical microscopy hundreds of cells flowing through the bed at physiological speeds. We find a large distribution of mobilities among red cells flowing through the structure; smaller channels provide a greater impedance to flow than larger ones, indicating that kinetic drag variations provide the origin of the distribution. The mobility of a particular cell is not correlated with the cell diameter but appears to be inversely correlated with intracellular calcium concentration of the cell, as determined by fluorescence of the calcium-binding dye fluo-3 AM. Also, we are able to use the parallel processing nature of our arrays to observe isolated events where the rigidity of the red cell seems to change suddenly over several orders of magnitude as it blocks a channel in the array.     

Studies of the deformability of preserved red blood cells using a simple filtration method]

The applied method to measure the filtrability of red blood cells is accurate enough for the study of the effects of the conditions of storage on their deformability. In this work the standard deviation of the method, the mean biological deviation of the deformability of human red blood cells and their deformability in the course of storage in the ACD-AG-medium have been investigated. Considerable loss of deformability occurs towards the end of storage time of the cells, which is not reversible be restitution of ATP alone. The applicability of this method as a simple test of the vitatity of preserved erythrocytes if to be tested.     

Purification of a human plasma membrane glycoprotein from human red blood cells by affinity chromatography using a monoclonal antibody

Using the monoclonal antibody LICR-LON-Fib75.1 coupled to Sepharose as an affinity chromatography column, a membrane glycoprotein with an apparent molecular weight of 18,000 on sodium dodecyl sulfate-polyacrylamide gels has been purified from human red blood cells. The purified protein contained 25% carbohydrate by weight, the predominant sugars being galactose, mannose, and glucosamine. Amino acid analysis indicated that the protein was relatively rich in aspartate, glutamate, valine, and leucine and had a low proline and methionine content. The molecule could be removed from intact red blood cells by trypsin and could be labeled with iodine by lactoperoxidase-catalyzed cell surface iodination of red blood cells. The protein could also be labeled using the lipidsoluble photoactivatable reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl) diazirine) and partitioned into the lower phase of the phase-separable detergent Triton X-114. During size-exclusion chromatography in different detergents alterations were observed in the apparent molecular weight of the protein. These results suggest that this Fib75.1-binding protein is an external red blood cell membrane glycoprotein which is capable of binding detergent. Proteins with a similar molecular weight have also been isolated from two human tumor cell lines by immunoprecipitation with this monoclonal antibody.