Cell membranes after malignant transformation. Part II: Dynamic stability with surface chemical reactions

The dynamic stability of cell membranes in presence of chemical reactions is analysed using the same hydrodynamic cell model as in Part I, with a spherical geometry. Chemical reactions give an additional contribution leading to instability even for positive total surface tension. The mechanical properties of the surface change drastically via the gradient of the surface tension (mechano-chemical coupling). An enzymatic regulation of cell division is proposed, via cAMP. Loss of contact-inhibition of division in cancer cells is interpreted as a lowering of the threshold for cell division, which is not modified at confluence. In that sense, failure of control mechanism in cancer cells is of more significance than rapid growth.     

Cell surface proteins and malignant transformation

Many of the altered properties of malignant cells are thought to involve alterations in cell surface functions. In order to understand these alterations it is necessary to know more about the molecular structure of the surface. Methods for analyzing surface proteins are discussed and their application to normal and transformed tissue culture cells are reviewed. A number of surface proteins are observed to be altered by transformation. Most of the alterations are reductions in amounts of particular species, although a few proteins do increase. Evidence concerning the reasons for these alterations and the possible functions of some of the molecules is reviewed. Working hypotheses arising from these data are presented and prospects for understanding the physiological changes in terms of molecular effects are discussed. Particular emphasis is placed on the idea that surface molecules are associated in specific-non-covalent complexes which are important for their functions.     

Increased trypsin sensitivity of cell surface macromolecules after malignant transformation.

The effect of treatment with 0.04% (w/v) trypsin (EC 3.4.4.4) for 3 h on the electrophoretic mobility (EPM) of polyoma-virus malignantly transformed BHK21 cells (Py6) and their normal counterparts has been investigated. These particular conditions were chosen because an earlier study had shown that such treatment released material from the Py6 cells which was not obtained from the BHK21 cells. The negative EPM of the Py6 cells at pH 7.5 was greatly increased by this treatment; whereas the EPM of the BHK21 cells remained unchanged. Active enzyme was required to produce the change. No evidence was obtained for cytolysis, cytotoxicity or uptake of the enzyme by the treated Py6 cells. Measurement of the EPM of the Py6 cells at different pH levels before and after trypsin treatment suggested that the enzyme was removing cationic groupings from the cell surface.     

Cell surface changes accompanying viral transformation: N-acetylneuraminic acid ectotransferase system activity.

The activity of the sialyl ectotransferase system of normal chick embryo fibroblasts (CEF) and chick embryo fibroblasts transformed with the Schmidt-Ruppin strain of Rous sarcoma virus (SR-RSV) have been compared. Neuraminidase treatment of the intact cells increased the sialyl ectotransferase system activity of control and transformed cells two to three times. The ectotransferase system activity increased as the pH was decreased from 7.8 to 6.0. The temperature optimum for both systems was 40 degrees C. Approximately 60% of the 14C-sialic acid incorporated at pH 6.5 or above could be removed with neuraminidase. The activity of the transformed cell system with or without neuraminidase treatment was more stimulated by addition of Mn2+ ions, particularly above pH 7.0. This difference in ion sensitivity indicates that a different cell surface phenomenon is being studied after transformation.     

Cell surface expression of v-fms-coded glycoproteins is required for transformation.

The viral oncogene v-fms encodes a transforming glycoprotein with in vitro tyrosine-specific protein kinase activity. Although most v-fms-coded molecules remain internally sequestered in transformed cells, a minor population of molecules is transported to the cell surface. An engineered deletion mutant lacking 348 base pairs of the 3.0-kilobase-pair v-fms gene encoded a polypeptide that was 15 kilodaltons smaller than the wild-type v-fms gene product. The in-frame deletion of 116 amino acids was adjacent to the transmembrane anchor peptide located near the middle of the predicted protein sequence and 432 amino acids from the carboxyl terminus. The mutant polypeptide acquired N-linked oligosaccharide chains, was proteolytically processed in a manner similar to the wild-type glycoprotein, and exhibited an associated tyrosine-specific protein kinase activity in vitro. However, the N-linked oligosaccharides of the mutant glycoprotein were not processed to complex carbohydrate chains, and the glycoprotein was not detected at the cell surface. Cells expressing high levels of the mutant glycoprotein did not undergo morphological transformation and did not form colonies in semisolid medium. The transforming activity of the v-fms gene product therefore appears to be mediated through target molecules on the plasma membrane.     

Proteolysis of chloroplast thylakoid membranes. I. Selective degradation of thylakoid pigment-protein complexes at the outer membrane surface

Isolated pea thylakoids were experimentally unstacked in low-salt buffer and incubated with Pronase or trypsin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that brief treatment with a very low concentration (1 μg/ml) of either enzyme had an effect primarily on the light-harvesting chlorophyll $\text{a}\text{b}$-protein complexes, which are more sensitive to proteolytic attack than the other proteins of the thylakoid membranes. This mild proteolysis cleaves a ~1000-dalton portion from the predominant 28,000-dalton polypeptide of these complexes. Extensive proteolysis (100 μg Pronase/ml for 15 min) degraded almost all membrane polypeptides not associated with the pigment-protein complexes and degraded the chlorophyll $\text{a}\text{b}$-protein complexes further than milder proteolysis. Pronase treatment of thylakoids in the presence of horseradish peroxidase was used to monitor membrane breakage during proteolysis. Treatment with 100 μg Pronase/ ml enabled considerable amounts of peroxidase activity, and presumably, proteolytic enzymes to enter into the intrathylakoid space. This trapping of peroxidase activity was seen only minimally with milder proteolysis (1 μg Pronase/ml). These results suggest that brief exposure to low concentrations of proteolytic enzymes affects only the outer, stromal thylakoid surface, while at higher concentrations, significant proteolysis takes place at both sides of the membrane.     

Cell surface changes during myoblast differentiation: preparation and carbohydrate composition of plasma membranes.

A technique for the preparation of plasma membrane from a skeletal muscle myogenic cell culture is described. Electron microscopic and enzymatic studies indicate that the preparation has kept its morphological integrity and has negligeable amount of cellular contiaminants. Carbohydrate composition studies have shown that the differentiated cells contain less hexosamine and sialic acid but accumulate glucose and galactose; the latter increase reflects the presence of a glucose-galactose-hydroxylysine unit which appears at the cell surface when myoblasts reach confluency.     

Cell culture and animal models of viral hepatitis. Part I: hepatitis B

Despite the existence of a preventative vaccine, HBV represents a substantial threat to public health, suggesting the need for research to develop new treatments to combat the disease. The authors review the available in vitro and in vivo models, including recently developed transgenic and chimeric mouse models.     

Polysomes from winter rye seedlings grown at low temperature : I. Size class distribution, composition, and stability

We have studied the influence of growth at low temperature on size class distribution, stability and composition of leaf cytoplasmic polysomes from rye seedlings (Secale cereale, cv Puma) grown at 5°C and at 20°C. Leaves of seedlings grown at 5°C contain 2.7 times more cytoplasmic polysomes (expressed on a DNA basis) and the polysome size class distribution is skewed toward larger polysomes. These changes were more pronounced in the free polysome fraction than in the membrane-bound fraction. The melting point of the total ribosome fraction from cold-grown leaves was decreased by 3.7°C. Electrophoresis did not reveal any difference in the rRNA or in core-ribosomal proteins (KCl nondissociable) following growth at low temperature. Some differences were noted in peripheral ribosomal proteins. This study is the first to examine the effect of growth at low and high temperatures on polysome metabolism using plants of similar developmental stage. Polysome quantity, polymerization, melting point and peripheral ribosomal proteins in rye seedlings are modified during growth at low temperature.     

Cell surface proteins of Drosophila: I. Changes induced by 20-hydroxyecdysone

Treatment of several Drosophila cell lines with the molting hormone (20-hydroxyecdysone) resulted in biochemical and cellular changes including the morphogenetic process of cell aggregation. Radiolabeling of the cell surface proteins revealed 34 polypeptides that are modulated by the hormone's action. This modulation included both expression of “new” proteins and disappearance of preexisting polypeptides. Whereas most of the hormone-induced proteins were lentil lectin-binding glycoproteins, only one group of disappearing proteins appears to bind lentil lectin. Labeling of the cell surface prior to hormone addition revealed no specific modification of preexisting surface proteins which could account for the protein changes observed with one possible exception. The potential relationship between the modulation in surface proteins and the increase in cell-cell adhesion that occurs during hormone exposure is discussed.     

Fluidized-bed denitrification for mine waters. Part I: low pH and temperature operation

Mining often leads to nitrate and metal contamination of groundwater and water bodies. Denitrification of acidic water was investigated in two up-flow fluidized-bed reactors (FBR) and using batch assays. Bacterial communities were enriched on ethanol plus nitrate in the FBRs. Initially, the effects of temperature, low-pH and ethanol/nitrate on denitrification were revealed. Batch assays showed that pH 4.8 was inhibitory to denitrification, whereas FBR characteristics permitted denitrification even at feed pH of 2.5 and at 7–8 °C. Nitrate and ethanol were removed and the feed pH was neutralized, provided that ethanol was supplied in excess to nitrate. Subsequently, Fe(II) and Cu impact on denitrification was investigated within batch tests at pH 7. Iron supplementation up to 100 mg/L resulted in iron oxidation and soluble concentrations ranging from 0.4 to 1.6 mg/L that stimulated denitrification. On the contrary, 0.7 mg/L of soluble Cu significantly slowed denitrification down resulting in about 45 % of inhibition in the first 8 h. Polymerase chain reaction—denaturant gradient gel electrophoresis demonstrated the co-existence of different denitrifying microbial consortia in FBRs. Dechloromonas denitrificans and Hydrogenophaga caeni were present in both FBRs and mainly responsible for nitrate reduction.     

Yeast alcohol dehydrogenase bound to membranes: surface and microenvironment effects on activity and stability.

The enzyme, yeast alcohol dehydrogenase, was adsorbed to porous nitrocellulose and nylon membranes. The two membranes provide different surface chemistries as indicated by the results of the streaming potential, enzyme adsorption, and fluorescein isothiocyanate adsorption experiments. The stability of the enzyme, as determined by continually measuring the extent of coenzyme reduction as a function of time, appeared to be much less for the enzyme adsorbed to the positively charged membrane surface. Moreover, the enzyme adsorbed to the positively charged membrane was the least responsive to pulses of the reducing agent, dithiothreitol, and appeared to exhibit the highest transition temperature when subjected to differential scanning calorimetry analysis. These results indicate that the entropically spreading process observed for other adsorbed proteins may be occurring and the process is more rapid and extensive when enzyme is adsorbed to the nylon than the nitrocellulose membrane. In addition to the relative stability of the enzyme on two different surfaces being examined, the effect of the microenvironment on modulating the activity of the enzyme was investigated by using the reversibility of the enzyme-catalyzed reaction as a probe of the average local environment of the enzyme. It was found that a threshold buffer concentration existed that, once exceeded, the effect of proton production by the reaction could be suppressed.     

Large-ligand adsorption to membranes: II. Disk-like ligands and shape-dependence at low saturation

Large-ligand adsorption to membranes or cells is considered in the absence of cooperative interactions. For the low-saturation regime, a general and exact treatment is given by means of the concept of excluded areas. With the help of this formalism, shape dependence of the adsorption behavior can be discussed quantitatively. In addition, a formalism is presented which allows to calculate binding curves at arbitrary saturation for ligands having a symmetric shape (disks, regular polygons). The underlying model is a modified version of the hard core fluid theory of Andrews (Andrews, F.C. (1976) J. Chem. Phys. 64, 1941–1947). Apart from applications to symmetric ligands, the results can be used to derive limiting conditions for ligands of any shape.     

Divalent cation-induced surface tension increase in acidic phospholipid membranes: Ion binding and membrane fusion

Chelation binding of divalent cations to phospholipid membranes may cause deformation in the headgroup regions of these lipid molecules. This deformation may be responsible for the observed large increase in surface tension of acidic phospholipid membranes induced by divalent cations. On the other hand, simple binding of monovalent cations without being followed by such a deformation of membrane molecules, does not result in a large surface tension increase in the membrane. A theoretical explanation for the above situation is given and the divalent cation-induced acidic phospholipid membrane fusion as well as other lipid membrane fusions are discussed in terms of the increased surface energy of membranes.     

Surface transformation of bioactive glass in bioreactors simulating microgravity conditions. Part I: experimental study

Surface modified bioactive glass with surface properties akin to those of the bone mineral phase is an attractive candidate for use as a microcarrier material for 3-D growth of bone-like tissue in rotating wall vessel bioreactors (RWVs). The critical surface properties of this material are the result of reaction in solution. Because an RWV environment is completely different from conditions previously employed for bioactive glass testing, a detailed study of the surface reactions is warranted. Under properly chosen conditions, RWVs can also provide a simulated microgravity environment for the bioactive glass (BG) particles. In this sense, this study is also a report on the behavior of a bioactive material under microgravity conditions simulated on earth. A high aspect ratio vessel (HARV) and carefully selected experimental conditions enabled the simulation of microgravity in our laboratory. A complimentary numerical study was simultaneously conducted to ascertain the appropriateness of the experimental parameters (particle size, particle density, medium density, medium viscosity, and rotational speed) that ensure simulated microgravity conditions for the glass particles in the HARV. Physiological solutions (pH 7.4) with and without electrolytes, and also with serum proteins, were used to study the change in surface character resulting from simulated microgravity. Control tests at normal gravity, both static and dynamic, were also conducted. Solution and surface analyses revealed major effects of simulated microgravity. The rates of leaching of constituent ions (Si-, Ca-, and P-ions) were greatly increased in all solutions tested. The enhanced dissolution was followed by the enhanced formation of bone-like minerals at the BG surface. This enhancement is expected to affect adsorption of serum proteins and attachment molecules, which, in turn, may favorably affect bone cell adhesion and function. The findings of the study are important for the use of bioactive materials as microcarriers to generate and analyze 3-D bone-like tissue structures in bioreactors under microgravity conditions or otherwise. Copyright John Wiley & Sons, Inc.     

Mould deterioration of feeding-stuffs in relation to humidity of storage: Part I. The growth of moulds at low humidities

The literature relating to the growth of moulds at different humidities is surveyed.
Observations were made on the development of mould growth on six widely different types of feeding-stuffs for storage periods extending over some 3 1/2 years. The main factors controlling mould growth were found to be:
1. The relative humidity rather than the moisture content of the food.
2. The length of the storage period. Mould growth took place relatively quickly on all feeding-stuffs stored at 100-75% R-H. Below 75% R.H. mould growth, while not prevented, only developed after a very prolonged latent period. Mould development was observed on locust beans exposed to a humidity as low as 65 % after a latent period of over 2 years.
3. The balance and type of nutrients provided by the various feeding-stuffs was shown to influence the latent period as well as the extent of mould deterioration. This was confirmed by experiments on artificially dried grass fractions and by experiments on starch, protein and fibre mixtures.
4. The temperature of storage was found to affect the rate of mould development; mould growth developed sooner on samples stored at 22C. than at 15.5C.
5. The type of mould species present. At high humidities all mould species grew rapidly: at low humidities, only a few types were capable of growth.