Acid phosphatase activity of human gingival fibroblasts measured using a simultaneous coupling semi-permeable membrane technique

Summary Acid phosphatase activity has been measured in cultured human gingival fibroblasts using a validated histochemical simultaneous coupling semi-permeable membrane technique. The histochemical reaction was linear over a three hour incubation period and had a pH optimum of 5.0. The activity was not increased by prior exposure to hypotonic acetate buffer and was inhibited by fluoride and molybdate but not by formaldehyde. These results indicate that the semi-permeable membrane technique described may be used for observing and measuring acid phosphatase activity in cultured fibroblasts. From results obtained using inhibitors, it appears that in these cells most of the acid phosphatase observed is lysosomal. The absence of any activation of activity following pre-incubation with hypotonic buffer indicates that the method is not suitable for monitoring lysosomal membrane function.     

Solution flow in tubular semi-permeable membranes

Summary A recent suggestion that the term volume flow should be used for the flow along sieve tubes when the pressure gradient involved is very small is considered to be unwarranted. It involves no new concepts and does not constitute any mechanism that is not implicit in the Münch theory.     

RNA cytometry of single-cells using semi-permeable microcapsules

Analytical tools for gene expression profiling of individual cells are critical for studying complex biological systems. However, the techniques enabling rapid measurements of gene expression on thousands of single-cells are lacking. Here, we report a high-throughput RNA cytometry for digital profiling of single-cells isolated in liquid droplets enveloped by a thin semi-permeable membrane (microcapsules). Due to the selective permeability of the membrane, the desirable enzymes and reagents can be loaded, or replaced, in the microcapsule at any given step by simply changing the reaction buffer in which the microcapsules are dispersed. Therefore, complex molecular biology workflows can be readily adapted to conduct nucleic acid analysis on encapsulated mammalian cells, or other biological species. The microcapsules support sequential multi-step enzymatic reactions and remain intact under different biochemical conditions, freezing, thawing, and thermocycling. Combining microcapsules with conventional FACS provides a high-throughput approach for conducting RNA cytometry of individual cells based on their digital gene expression signature.     

Purification of antibiotics by ultrafiltration through semi-permeable membranes

The process of purification of benzylpenicillin, penicillin V and erythromycin solutions with the use of semi-permeable membranes was studied. Practically complete separation of high-molecular admixtures and microbial colonies and separation of significant amounts of the colored compounds were shown to be possible. The purification process may be used effectively at the early stages of antibiotic production.     

Mean residence time of molecules diffusing in a cell bounded by a semi-permeable membrane: Monte Carlo simulations and an expression relating membrane transition probability to permeability

 The rapid exchange of water across erythrocyte membranes is readily measured using an NMR method that entails doping a suspension of cells with a moderately high concentration of Mn²⁺ and measuring the rate of transverse relaxation of the nuclear magnetisation. Analysis of the data yields an estimate of the rate constant for membrane transport, from which the membrane permeability can be determined. It is assumed in the analysis that the efflux rate of the water is solely a function of the rate of membrane permeation and that the time it takes for intracellular water molecules to diffuse to the membrane is relatively insignificant. The limits of this assumption were explored by using random-walk simulations of diffusion in cells modelled as parallel planes, spheres, and biconcave discs. The rate of membrane transport was specified in terms of a transition probability but it was not initially clear what the relationship should be between this parameter and the diffusional membrane permeability P _d. This relationship was derived and used to show that the mean residence time for a water molecule is determined by P _d when the diffusion coefficient is above a certain threshold value; it is determined by the distance to the membrane below that value. Received: 7 January 2000 / Revised version: 4 April 2000 / Accepted: 4 April 2000     

Models of delta-hemolysin membrane channels and crystal structures

Molecular modeling and energy calculations have been used to study how delta-hemolysin and melittin helices may aggregate on membrane surfaces and insert through membranes to form channels. In these models adjacent antiparallel amphipathic helices form planar "raft" structures, in which one surface is hydrophobic and the other hydrophilic. Models of delta-hemolysin crystal structure were developed using these "rafts." These models are based on the unit cell constants and the crystal symmetry obtained from the preliminary crystal data. Energy calculations favor channel models of delta-hemolysin with six or eight monomers per channel.     

Nowhere to run: semi-permeable barriers affect pronghorn space use

Animal movement can mediate the ecological consequences of fragmentation; however, barriers such as fences, roads, and railways are becoming a pervasive threat to wildlife. Pronghorn (Antilocapra americana) habitat in western North America has been fragmented by roads, railways, and fences. Although pronghorn are sensitive to barriers, neither the relative permeability of different barriers to crossing nor their influence on space use have been quantified. We used a large global positioning system (GPS)-collar dataset of pronghorn (n = 1,010 animal-years) in Wyoming, USA, to first quantify the likelihood that pronghorn cross each of 5 different anthropogenic barriers, including fences, county roads, railroads, state highways, and interstate highways (i.e., interstates). Next, we assessed how each barrier influenced pronghorn space use during the winter as indexed by the area occupied, and daily displacement relative to the density of barriers on an individual's winter range. The semi-permeability of the 5 barriers varied substantially, with the interstate being the most severe barrier to pronghorn movement. Pronghorn were >300 times less likely to cross interstates compared to state highways. Although pronghorn space use was rarely influenced by barriers within individual core winter ranges, pronghorn space use was constrained by barriers on the buffered periphery of individual winter ranges. Despite their different permeability to movement, the density of fences and combined interstates and railroads had similarly negative effects on pronghorn space use. Our results illustrate that the degree to which pronghorn avoid crossing barriers may scale up to affect access to habitat. Additionally, our results indicate that the effects of barriers on habitat access are not proportional to their permeability. Our results add to a growing consensus that effective management of mobile species depends on understanding how different kinds of semi-permeable barriers influence access and use of habitats.     

Immobilization of chloroperoxidase from Serratia marcescens in semi-permeable protein membranes

A method for the immobilization of chloroperoxidase from the bacterial strain Serratia marcescens in semi-permeable membranes was developed based on the following proteins: bovine serum albumin, gelatin, ribonuclease, cytochrom C, and the protein of the covering layer of Bacillus sphaericus cells. Estimation of the activity and stability of immobilized preparations in a batch reactor was carried out. Published in Russian in Prikladnaya Biokhimiya i Mikrobiologiya, 2006, Vol. 42, No. 2, pp. 152–155. This article was translated by the authors.     

Features critical for membrane binding revealed by DivIVA crystal structure

DivIVA is a conserved protein in Gram‐positive bacteria that localizes at the poles and division sites, presumably through direct sensing of membrane curvature. DivIVA functions as a scaffold and is vital for septum site selection during vegetative growth and chromosome anchoring during sporulation. DivIVA deletion causes filamentous growth in Bacillus subtilis, whereas overexpression causes hyphal branching in Streptomyces coelicolor. We have determined the crystal structure of the N‐terminal (Nt) domain of DivIVA, and show that it forms a parallel coiled‐coil. It is capped with two unique crossed and intertwined loops, exposing hydrophobic and positively charged residues that we show here are essential for membrane binding. An intragenic suppressor introducing a positive charge restores membrane binding after mutating the hydrophobic residues. We propose that the hydrophobic residues insert into the membrane and that the positively charged residues bind to the membrane surface. A low‐resolution crystal structure of the C‐terminal (Ct) domain displays a curved tetramer made from two parallel coiled‐coils. The Nt and Ct parts were then merged into a model of the full length, 30 nm long DivIVA protein.     

Normalizing sweet orange (C. sinensis (L.) osbeck) somatic embryogenesis with semi-permeable membranes

Summary Development of citrus somatic embryos initiated from embryogenic callus generally results in abnormal morphogenesis of somatic embryos. To normalize development, glycerol-induced globular-stage somatic embryos of sweet orange [C. sinensis (L.) Osbeck cv. ‘Hamlin’] were cultured on 6000–8000 MW cutoff cellulose acetate, >400 000 MW cutoff cellulose acetate, nitrocellulose, polyvinylidene fluoride (PVDF), cellulose filter paper, or positive or neutral charged nylon membranes. Only the two cellulose acetate membranes resulted in the development of normal, two-cotyledon, bipolar, heart-shaped embryos, and no aberrant teratoma-like structures. Heart-shaped embryos developed and germinated normally on Murashige and Tucker basal medium with 0.5% sucrose and 1 μM gibberellic acid. Culture of embryogenic callus directly onto cellulose membranes also resulted in the development of normal heart-shaped embryos, indicating that glycerol induction of globular-stage embryos is not necessary. Heart-shaped embryos were not observed when the osmotic potential of the medium was increased by the addition of 2.5–15% polyethylene glycol; neither were they observed when the matric potential of the medium was increased by increasing the gelling agent concentrations of agar and Gelrite from 0.8% to 3% and 0.15% to 0.9%, respectively.     

Immobilization of chloroperoxidase from Serratia marcescens in protein semi-permeable membranes

A method for the immobilization of chloroperoxidase from the bacterial strain Serratia marcescens in semi-permeable membranes was developed based on the following proteins: bovine serum albumin, gelatin, ribonuclease, cytochrom C, and the protein of the covering layer of Bacillus sphaericus cells. Estimation of the activity and stability of immobilized preparations in a batch reactor was carried out.