A novel technique for entrapment of hybridoma cells in synthetic thermally reversible polymers

Summary A new technique for entrapment of cells in thermally reversible polymer (poly-N-vinylcaprolactam - PVCl) has been developed. Using stabilizers, such as ovalbumin, sodium carboxymethylcellulose or egg powder were necessary to give structures that were stable upon stirring carriers. Two hybridoma lines, in particular, 3B4 and 1A10 cells were entrapped in gel and produced monoclonal antibodies IgG and IgM classes, respectively, against Arabis mosaic virus.     

Use of PVA-cryogel entrappedCitrobacter intermedius cells for continuous production of 3-fluoro-L-tyrosine

Summary The cells ofCitrobacter intermedius (containing L-tyrosine phenol lyase which catalyses the synthesis of 3-fluoro-L-tyrosine fromo-fluorophenol, pyruvate and ammonia) were entrapped into 10% poly(vinyl alcohol) cryogel beads. Cryogenic immobilization resulted in a moderate increase of the synthetic activity of the cells. During continuous operation for one month in a packed bed column reactor the productivity of the immobilized biocatalyst decreased by 30%.     

Materials on the serologic study of children inoculated with live measles vaccine

The results of serological examination of children, residents of Leningrad, vaccinated with live measles vaccine, are presented. A total of 2012 children were examined. Antibodies were absent in children of different age in 4.2--15.8% of cases. The greatest percentage of secronegative children (15) was noted among those vaccinated at the age of under one year. The mean geometrical antibody titres were the greatest in children aged from 7 to 14 years. The values of these mean titres were less in children vaccinated at the age of under one year than in those vaccinated later--6.5--8.6 and 10.6--11.3, respectively.     

The development of methods for obtaining monospecific ingredients for immunoenzyme analysis

The methods of the modification of Salmonella O- and H-antigens and the preparation of biologically active sorbents on their basis have been developed. The use of these sorbents has permitted the isolation of affinity antibodies with strictly defined specific activity. The work shows the possibility of the successful use of carriers obtained on the basis of porous glass, chemically modified by acrylic copolymers containing activated carboxylic groups, and intended for the immobilization of antigens of both protein and carbohydrate nature.     

Aconitine modification of cell membrane sodium channels of a neuroblastoma

Currents through normal and aconitine-modified sodium channels in the perfused neuroblastoma cell are measured under voltage clamp conditions. Aconitine shifts the voltage range of activation of the sodium channels towards more negative potentials by about 20 mV, and changes the selectivity, so that channels become more permeable to NH4+ than to Na+ ions. The currents through aconitine--modified channels are inactivated almost completely like those through the normal ones. Aconitine is effective when applied to both sides of the cell membrane. Steady-state characteristics of gating are discussed in terms of the model assuming three main states of the gate machinery: closed, open and inactivated.     

The store-operated calcium entry pathways in human carcinoma A431 cells: functional properties and activation mechanisms

Activation of phospholipase C (PLC)-mediated signaling pathways in nonexcitable cells causes the release of Ca2+ from intracellular Ca2+ stores and activation of Ca2+ influx across the plasma membrane. Two types of Ca2+ channels, highly Ca2+-selective ICRAC and moderately Ca2+-selective ISOC, support store-operated Ca2+ entry process. In previous patch-clamp experiments with a human carcinoma A431 cell line we described store-operated Imin/ICRACL plasma membrane Ca2+ influx channels. In the present paper we use whole-cell and single-channel recordings to further characterize store-operated Ca2+ influx pathways in A431 cells. We discovered that (a) ICRAC and ISOC are present in A431 cells; (b) ICRAC currents are highly selective for divalent cations and fully activate within 150 s after initiation of Ca2+ store depletion; (c) ISOC currents are moderately selective for divalent cations (PBa/PCs = 14.5) and require at least 300 s for full activation; (d) ICRAC and ISOC currents are activated by PLC-coupled receptor agonists; (e) ISOC currents are supported by Imin/ICRACL channels that display 8.5-10 pS conductance for sodium; (f) ICRAC single channel conductance for sodium is estimated at 0.9 pS by the noise analysis; (g) Imin/ICRACL channels are activated in excised patches by an amino-terminal fragment of InsP3R1 (InsP3R1N); and (h) InsP3 binding to InsP3R1N is necessary for activation of Imin/ICRACL channels. Our findings provide novel information about store-operated Ca2+ influx pathways in A431 cells.     

Immobilization of proteases in composite hydrogel based on poly(N-vinyl caprolactam)

Summary A novel one-step method was developed for entrapment of proteolytic enzymes, namely carboxypeptidase B and trypsin, in polymer composite gel based on poly(N-vinyl caprolactam). Native proteases and enzymes previously stabilized by covalent attachment to poly(vinylpyrrolidone-co-acrolein) were immobilized in gels. After immobilization, about 90% and 75% of original trypsin and CPB activities, respectively, were retained. The immobilized enzymes were active within a wide pH range. The optimum temperature of the entrapped enzymes was approximately 25°C higher than that of the soluble enzymes. The entrapped enzymes were successfully used to obtain human insulin from recombinant proinsulin.     

Temperature and pH sensitivity of the O640 intermediate of the bacteriorhodopsin photocycle

The temperature and pH dependencies of the O₆₄₀ intermediate of the photocycle of bacteriorhodopsin (bR) were investigated by flash photolysis and T-jump experiments. The maximal concentration of the O₆₄₀ intermediate was found to be dependent on the temperature, which is described by a sigmoidal relationship. With increasing pH the midpoint of the sigmoidal curves shifts to higher temperatures. The Van't Hoff equation provides enthalpy and entropy values of the observed states. These results indicate that, in the investigated temperature (0-60°C) and pH (pH 4.0-10.0) range, the sequence of the principal intermediates in the pathway “M-N-O-bR” does not change. The observations of the O₆₄₀ intermediate at pH < 8.0 and of the N₅₅₀ intermediate at pH > 8.0 are most probably due only to changes of the intrinsic rate constants of the bR photocycle, not to a different mechanism.     

A study of moss (Marchantia polymorpha) thylakoid membrane lipids in monolayers.

Monolayers of seven fractions of natural lipids (phosphatidyl inositol, sulfoquinovosyl diacylglycerol, phosphatidylcholine, digalactosyl diacylglycerol, phosphatidyl glycerol, phosphatidylethanolamine, monogalactosyl diacylglycerol), isolated from the photoautotrophic cell culture of the moss Marchantia polymorpha grown under normal and light-stress conditions, have been prepared for the first time. We have shown that the high-intensity light affects the area occupied by the lipid molecule. In the case of digalactosyl diacylglycerol and phosphatidyl glycerol fractions, after the light stress the area significantly increased from 0.50 to 0.80 nm2 and from 0.47 to 0.63 nm2, respectively, and in the case of the sulfoquinovosyl diacylglycerol fraction, the area decreased from 0.40 to 0.32 nm2. These results are in agreement with our previous data on the redistribution of the double bonds in the aliphatic chains of these lipids and can be used to characterize the state of the lipid bilayer of the thylakoid membranes.