Cellophane Membrane Research Articles

Electrophoretic immunodesorption of proteins according to molecular weight by use of a double-membrane system

A simple method is described for electrophoretic desorption of proteins from antigen-antibody complexes, with more than 90% recovery and without denaturation, after immunosorbent affinity chromatography. Radiolabeled or unlabeled human serum albumin (HSA) and α-1-antitrypsin (AAT), conjugated to rabbit anti-HSA or anti-AAT polyclonal antisera, respectively, were electrophoretically desorbed from Sepharose 4B. In addition, purification and concentration of the major HSA protein band (monomer) of 68 kD from the other oligomeric protein bands were achieved by use of a two-membrane system in a simple electroelution apparatus. The system consisted of an upper cellulose acetate membrane, with pore size 20 nm and separation limit 70 kD, and a lower dialysis cellophane membrane with molecular weight cut-off from 1–50 kD that cnables separation according to size. Furthermore, purification of the monomer HSA or AAT from normal human serum was performed with 92% recovery. Homogeneity was implied by the presence of one band after sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, Western blot, and autoradiography.

The auxin transport inhibitor 2,3,5-triiodobenzoic acid (TIBA) inhibits the stimulation of in vitro lateral root formation and the colonization of the tap-root cortex of Norway spruce (Picea abies) seedlings by the ectomycorrhizal fungus Laccaria bicolor.

Norway spruce (Picea abies (L.) Karst.) seedlings were inoculated with the ectomycorrhizal fungus Laccaria bicolor ((Marie) Orton), strain S238 N, in axenic conditions. The presence of the fungus slowed tap-root elongation by 26% during the first 15 d after inoculation and then stimulated it by 136%. In addition, it multiplied in vitro lateral root formation by 4.3, the epicotyl growth of the seedlings by 8.4 and the number of needles by 2. These effects were maintained when the fungus was separated from the roots by a cellophane membrane preventing symbiosis establishment, thus suggesting that the fungus acted by non-nutritional effects. We tested the hypothesis that IAA produced by L. bicolor S238 N would be responsible for the stimulation of fungal induced rhizogenesis. We showed in previous work that L. bicolor S238 N can synthesize IAA in pure culture. Exogenous IAA supplies (100 and 500 μm) reproduced the stimulating effect of the fungus on root branching but inhibited root elongation. The presence of 2,3,5-triiodobenzoic acid (TIBA) in the culture medium significantly depressed lateral root formation of inoculated seedlings. As TIBA had no significant effect on IAA released in the medium by L. bicolor S238 N, but counteracted the stimulation of lateral rhizogenesis induced by an exogenous supply of IAA, we suggest that TIBA inhibited the transport of fungal IAA in the root. Furthermore TIBA blocked the colonization of the main root cortex by L. bicolor S238 N and the formation of the Hartig net. These results specified the role of fungal IAA in the stimulation of lateral rhizogenesis and in ectomycorrhizal symbiosis establishment.

The expression of genes involved in parasitism by Trichoderma harzianum is triggered by a diffusible factor.

The mycoparasite Trichoderma harzianum has been extensively used in the biocontrol of a wide range of phytopathogenic fungi. Hydrolytic enzymes secreted by the parasite have been directly implicated in the lysis of the host. Dual cultures of Trichoderma and a host, with and without contact, were used as means to study the mycoparasitic response in Trichoderma. Northern analysis showed high-level expression of genes encoding a proteinase (prb1) and an endochitinase (ech42) in dual cultures even if contact with the host was prevented by using cellophane membranes. Neither gene was induced during the interaction of Trichoderma with lectin-coated nylon fibres, which are known to induce hyphal coiling and appressorium formation. Thus, the signal involved in triggering the production of these hydrolytic enzymes by T. harzianum during the parasitic response is independent of the recognition mediated by this lectin-carbohydrate interaction. The results showed that induction of prb1 and ech42 is contact-independent, and a diffusible molecule produced by the host is the signal that triggers expression of both genes in vivo. Furthermore, a molecule that is resistant to heat and protease treatment, obtained from Rhizoctonia solani cell walls induces expression of both genes. Thus, this molecule is involved in the regulation of the expression of hydrolytic enzymes during mycoparasitism by T. harzianum.

Target Site of a New Antifungal Compound KC10017 in the Melanin Biosynthesis ofMagnaporthe grisea

A new experimental fungicide KC10017 (3-[4′-bromo-2′, 6′-dimethylphenoxy]methyl-4-[(3″-methylphenyl) aminocarbonyl]methyl-1,2,4-oxadiazol-5-one) completely controlled rice blast disease at more than 1.0 μg/ml without curative activity. When the chemical was applied after the inoculation was made with injury, the fungicide had no effect on controlling rice blast. The fungicide at concentrations of more than 1 μg/ml blocked appressorial melanization and penetration of cellophane membrane byMagnaporthe griseaP-2 without affecting fungal growth, spore germination, and appressorial formation. To elucidate the target site of KC10017 in melanin biosynthesis inhibition,M. griseaP-2 was grown in potato dextrose broths treated with both KC10017 and tricyclazole. The mycelia and culture media of cultures treated with both KC10017 and tricyclazole at a concentration of 5 μg/ml each became colorless like as the cultures treated with KC10017 alone, whereas the tricyclazole-treated cultures exhibited red color. Shunt products of 3,4-dihydro-3,4,8-trihydroxy-1-(2H)-naphthalenone, 3,4-dihydro-4,8-dihydroxy-1-(2H)-naphthalenone, 3,4-dihydro-4,6,8-trihydroxy-1-(2H)-naphthalenone, and 4-hydroxyscytalone were absent or present in only trace amounts in the cultures treated with KC10017 alone or both KC10017 and tricyclazole. However, they were detected at much higher levels in the cultures treated with tricyclazole alone. On the other hand, appressorial melanization and penetration ability inM. griseatreated with KC10017 was recovered by the addition of scytalone. These results demonstrate that the target site of KC10017 in melanin biosynthesis inhibition is at the pentaketide synthesis step and/or at the pentaketide cyclization step prior to 1,3,6,8-tetrahydroxynaphthalene formation.

MOLECULAR STRUCTURAL PARAMETERS IN TUBERCULOSTATIC ACTIVITY OF POLYHYDROXYXANTHONES

This investigation was undertaken to test the correlation of the observed tuberculostatic activity of a series of 7(2)-polyhydroxyxanthones 1a-j with their lipophilic parameters (Rm) as well as molecular orbital descriptors such as; dipole moment (dp); electro density (D); heat of formation (hf), torsion angle (tor); van der Waal forces (vdw), bond length (str) and bond angle (bnd). The Rm-values were calculated from the corresponding Rf­-values, determined using polyamide plates. The molecular orbital regressors were derived through semi-empirical calculations using MMX-computer program. QSAR analysis of the activity parameters (Ka, Log Ka, Ka* and logKa*) as dependent variables and Rm or combination of Rm and dummy parameters representing the substitution pattern of hydroxyl groups on ring C of the xanthone nucleus demonstrated an increase in the lipophilic character by itself which did not correlate (r» -0.42) with increased potency against Mycobacterium lufu. Permeability study using standard cellophane membrane showed that, the synthesized compounds were poorly penetrating and may act directly on the cell wall. On the contrary higher correlation (r= 0.91) was observed between the activity parameter (log Ka*) and the electron density at C-4.

IV -VITRO AND IN -VIVO AVAILABILITY OF NAPROXEN FROM TRANSPARENT OIL/ WATER GELS IN COMPARISON TO SOME CONVENTIONAL TOPICAL BASES

Within the scope of evaluating transparent oil/water (TOW) gels as dermatological vehicles, the anti-inflammatory and analgesic agent Naproxen (NA) was incorporated in a transparent oil-water gel containing two emulsifying agents (Emulgin B3 and Cetiol HE), an oily liquid (isopropylpalmitate) and water. The release rate of NA from the TOW gel formulation through a cellophane membrane into phosphate buffer (pH 6) at 37°C was studied. The percutaneous absorption in rabbits was evaluated and compared with the absorption from a hydrogel and from a hydrophilic base formulation. The area under the curve and the Cmax values in plasma were significantly higher for the TOW gels in comparison with other formulations after single application. A good correlation was observed between the in-vitro and in-vivo data.

Influence of sucrose, mucin and xanthan gum on spore germination of ten different fungi

Germination of conidia, uredospores and basidiospores of ten diverse fungi were compared in sterile distilled water, 0.5% sucrose, 0.5% mucin and 0.5% xanthan gum. All three compounds generally increased spore germination on cellophane membranes compared to water, but mucin was the most effective. Mucin stimulated significantly more germination than sucrose for 7 of the 10 fungi, and only Aspergillus niger and Colletotrichum gloeosporioides showed greater spore germination in sucrose than in mucin. Sucrose had no effect on uredospore or basidiospore germination, but mucin stimulated germination of these spores. Mucin was more effective than xanthan gum in increasing spore germination for 9 of the 10 fungi. Animal mucin is chemically similar to the natural spore matrix of some fungi and may provide some of the benefits of the spore matrix without the drawback of possible germination inhibitors. Animal mucin may be a useful substitute for the spore matrix in inoculations of a variety of fungi.

Development of Leptographium wageneri on root surfaces and other substrata

SummaryConidia of Leptographium wageneri var. ponderosum, Leptographium wageneri var. pseudotsugae and Leptographium wageneri var. wageneri germinated on wounded roots of ponderosa pine (Pinus ponderosa), Douglas fir (Pseudotsuga menziesii), and pinyon (Pinus edulis), but not on unwounded roots. On wounds, germination occurred regardless of the combination of pathogen variety and plant species. When wounded roots of ponderosa pine and Douglas fir seedlings were inoculated with conidia of L. w. var. ponderosum and L. w. var. pseudotsugae, respectively, 87‐100% of the seedlings became infected. When unwounded roots were inoculated with wooden blocks colonized with L. wageneri, 60‐66% of the seedlings became infected.The development of L. w. var. ponderosum and L. w. var. pseudotsugae on agar was compared with their development on agar overlaid with either cellophane or Nucleopore membranes. Undulating hyphae were found commonly in and on the agar, but less frequently on the membranes. On both membranes, mycelia formed mats which were tightly appressed to the surface; mats were not formed on the agar. On both agar and the membranes, two or more hyphae often formed a strand. When L. w. var. ponderosum mycelia were allowed to colonize the surface of an unwounded pine root, strands, undulating hyphae and poorly formed mats were seen.

Acrylic type polymers containing ibuprofen and indomethacin with difunctional spacer group: synthesis and hydrolysis

Acryloyl- and methacryloyloxyethyl ester and amide of ibuprofen and indomethacin, two kinds of non-steroidal anti-inflammatory drugs, were prepared under mild conditions. A difunctional spacer group was introduced between the drug and acrylic moiety of the monomer through an hydrolyzable ester or amide linkage. Free radical polymerization of the resulting monomers were carried out in DMF, DMSO or dioxane solution, using AIBN as initiator at the temperature range 60–70°C. The polymers were characterized by FT-IR, 1H NMR and 13C NMR spectroscopies. Stereochemical configuration of monomeric units along the polymer chain had been analysed by NMR spectroscopy. Gel permeation chromatography (GPC) was used for determination of average molecular weights of drug pendent polymers. Glass transition temperature (Tg) of the polymers was determined calorimetrically. The hydrolysis of drug-polymer conjugates was carried out in cellophane membrane dialysis bags containing aqueous buffer solutions (pH 8 and pH 1) at 37°C. Detection of hydrolysis solution by UV spectroscopy showed that the drug can be released by hydrolysis of ester or amide bonds between the drug and spacer group. The acryloyloxyethyl type polymers having a polar amide group were hydrolyzed rather easily.

Capillary zone electrophoresis in a hydrodynamically closed separation system with enhanced sample loadability

Some phenomena linked with capillary zone electrophoresis (CZE) performed in a hydrodynamically closed separation system with enhanced sample load capacity via the use of capillary tubes of larger I.D.s were studied. Calculations of the plate heights for varying amounts of the analytes loaded onto 50 and 300 μm I.D. columns under identical CZE separating conditions revealed that the analytical advantages of using columns of larger I.D. include significantly reduced contributions of electromigration dispersion. At higher concentrations of the carrier electrolytes this gain, however, can be partially lost due to increased thermal dispersive effects. Calculated resolutions for a varying ratio of a pair of the analytes loaded onto 50 and 300 μm I.D. columns favoured the use of the latter I.D. in situations when the ratio of the analytes was higher than ca. 102:1. CZE experiments were carried out in a 300 μm I.D. capillary tube made of fluorinated ethylene–propylene copolymer (FEP) with a porous cellophane membrane serving as a hydrodynamic barrier to prevent a flow of the solution in the separation compartment due to a pressure difference between the electrode vessels. Movement of the membrane was found to be a source of undesired flows in the separation compartment, which adversely affected both the reproducibilities of migration times of the analytes and their separation efficiencies. Mechanical support eliminated these problems. Dispersive phenomena associated with electroosmosis in the closed separation compartment were effectively suppressed by using high molecular weight derivatives of water soluble polymers (methylhydroxyethylcellulose and polyethyleneglycol) in the carrier electrolyte solutions. Examples from the separations of various groups of analytes (synthetic food colourants, some inorganic anions, alkali and alkaline earth metal cations and glycoforms of erythropoietin) are given to illustrate a practical utility of the studied CZE approach.

Effect of Ultrasound on Driving Force of Diffusion Dialysis

In this paper, we discuss the effect of ultrasound on the driving force of diffusion dialysis which we demonstrate through experiments. The experiments were conducted using a cellophane membrane and 0.025 wt% NaCl solution at 25° C under ultrasonic irradiation of different sound pressures from 55 kPa to 100 kPa, at a frequency of 27.5 kHz. It was found that the amount of NaCl permeated decreases with increasing sound pressure when the diffusion direction is opposite to the irradiation direction of ultrasound.

Effect of Ultrasonic Irradiation on Permeability of Dialysis Membrane

The application of ultrasound for accelerating the diffusion of electrolytes through a membrane has been attempted. However, the effect of ultrasound on membrane permeability was not clarified in previous studies. we report the relationship between the time of ultrasound irradiation to membrane and permeability of the membrane, and the relationship between sound pressure and the membrane lifetime. Experiments were conducted using a cellophane membrane which had been irradiated for a certain time by ultrasound of different sound pressures at a frequency of 27.5 kHz, and the permeability of the membrane was examined with 1 wt% NaCl solution at 25° C. It was found that the membrane permeability increases with irradiation time. Furthermore, the lifetime of membrane decreases with increasing sound pressure. The degradation of the permeability by ultrasound is due to ultrasonic cavitation.

Isolation of Microbial Antagonists for Biocontrol of Grey Mould Disease of Strawberries

A screening programme is described for the assessment of the potential of biocontrol agents to control grey mould of strawberries caused by Botrytis cinerea. Bacteria were isolated from strawberry fruits, leaves and flowers from a commercial field site and screened for antagonism towards B. cinerea using two in vitro and one in vivo screening techniques. From 559 microorganisms isolated, 108 inhibited pathogen growth on agar plates and 27 of these prevented spore germination on Cellophane membranes. The ability of these 27 isolates to inhibit infection of young strawberry leaves by B. cinerea on whole plants under glass was then tested. Seven isolates reduced grey mould development and were subsequently assessed in a field trial. Two isolates, one of Bacillus pumilus and one of Pseudomonas fluorescens, were as effective or more effective than standard dichlofluanid sprays and may therefore be of potential value as antagonists of B. cinerea.

Effect of UV Light on Different Structural and Transport Parameters of Cellophane Membranes

A comparative study of UV light influence on structural and transport parameters of cellophane membranes was made. Changes in the chemical structure and electrical behavior of cellophane membranes were considered by determining the hydraulic permeability, salt diffusion coefficient, and resistance values, as well as some geometrical parameters, for an untreated membrane and two differently UV-treated cellophane membranes. Differences in the characteristic parameters for the three samples showed that radiation mainly affected the membrane structure, while only small changes in membrane electrical behavior were determined.

High resolution density gradient electrophoresis of cellular organelles.

A density gradient electrophoresis apparatus made of Perspex was constructed, with a separation column (7 x 2.2 cm) containing a 0-5% linear Ficoll gradient. The useful separation path is 6 cm. A specially designed gradient mixer is described which fits over the application cone. This cone permits precise gradient and sample introduction as well as undisturbed fractionation after electrophoresis. A bottom circular palladium cathode is separated hydrodynamically but not electrically from the density gradient by a cellophane membrane, merely secured by an O-ring. The top circular platinum anode allows for upward electrophoresis (80-100 min at 10 mA). The markedly higher resolution of subcellular organelles was compared with separations obtained earlier with a small, but much more difficult to fabricate, prototype. Moreover, ease of manipulation was greatly improved. A wide separation distance was obtained between plasma membrane, endoplasmatic reticulum as well as between two populations of lysosomes. Even early, middle, and late endosomes could be separated with high resolution. Soluble isoenzymes could be separated as well and were far away from the vesicle-enclosed enzymes.

An Apparatus (Georgiou-Petri Dish) for Growing Fungi and other Microorganisms on Liquid Media in a Petri Dish

This work describes a new apparatus for growing fungi and other microorganisms on liquid nutrient media in a Petri dish. The apparatus is composed of a net supporting a cellophane membrane stretched between an outer and an inner ring that is placed inside a Petri dish. This modification of the standard Petri dish offers many advantages for studying growth, metabolism, differentiation, and other aspects of fungi in liquid cultures with minimal waste of expensive chemicals. Monitoring of excreted or absorbed substances by the fungi, the aseptic transfer of undisturbed fungal colonies from dish to dish, and harvesting are made easier, using this apparatus.

Method of determination of cellulase activity in soils and in microbial cultures, and its calibration

A new method has been developed for determining cellulase activity (CA) in soils and in microbial cultures. This procedure is based on the measurement of the loss in strength of a cellophane membrane (Proc. USSR Acad. Sci. Ser. Biol. 3 (1988) 466–471) and has been calibrated with cellulase enzyme activity, so that it is now possible to relate the decline in membrane strength to units of cellulase enzyme. The calibration curve was used to convert the data obtained in relative units (atm h −1) to standard enzymatic units (μg reduced sugars (red.sug.) ml −1 h −1). Using group-specific antibiotics: actidione and chloramphenicol the method was applied to estimate the differential contribution of soil fungi, bacteria and extracellular enzymes to cellulose decomposition in soil. The CA was measured in: pure cultures of fungi, Trichocladium asperum and Acremonium charticola (from 0.106 to 0.319 μg red.sug. ml −1 h −1), soil samples (0.158 and 0.176 μg red.sug. ml −1 h −1) and in situ, for a vertical profile (5–30 cm) of an oligotrophic bog (0.058-0.005 μg red.sug. ml −1 h −1), and in several watershed forest soils (from 0.158 to 0.206 μg red.sug. ml −1 h −1).

Comparison of biomass dry weights and radial growth rates of fungal colonies on media solidified with different gelling compounds

Penicillium commune, Aureobasidium pullulans, and Paecilomyces farinosus were grown on two different media solidified with agar, Pluronic F-127, Carrageenan X-4910, or Carrageenan X-4910 overlaid with cellophane. Growth on Carrageenan X-4910 was generally the same as that on agar, as was the visual appearance of the colonies, e.g., the pigmentation. The Carrageenan X-4910 gels had a melting point, depending on the medium, of 41 to 46(deg)C, and the dry weights of the colonies were readily determined at 60(deg)C. To determine the dry weights of the colonies grown on agar plates, the gels were boiled for 10 min to melt the agar. Comparison of these two procedures showed that the boiling procedure resulted in a 22% reduction of the biomass dry weight. Cellophane membranes did not affect the radial growth rate profoundly. The biomass density was almost halved for P. commune and P. farinosus grown with membranes, whereas the presence of the membrane did not affect the biomass density of A. pullulans. The biomass densities of the colonies grown on Pluronic F-127 were significantly reduced, while in most cases, the radial growth rates of colonies grown on Pluronic F-127 were significantly higher than those obtained on agar or Carrageenan X-4910. Furthermore, the morphology of the leading hyphae was altered, and the hyphal growth unit length was more than twice that obtained on agar and Carrageenan X-4910. Carrageenan X-4910 is a valuable gelling compound for the study of the growth of fungi, as the biomass dry weight is readily determined and growth is similar to that obtained on agar gels.

Enhancement in Diffusion of Electrolyte through Membrane Using Ultrasonic Dialysis Equipment with Plane Membrane

Application of ultrasound to accelerate the dialysis separation of electrolytes through a membrane was studied with ultrasonic dialysis equipment. The experiments were conducted with cellophane membrane and KCl solution, CH3COONa solution, and a mixture of KCl and saponin solutions. It was found that the diffusion velocity of electrolyte through a membrane with ultrasonic irradiation is faster than that without ultrasonic irradiation, and it increases with acoustic pressure. It has become clear that the reasons for enhancement caused by ultrasound are increase in liquid particle velocity and diffusion coefficient due to ultrasonic vibration. It was confirmed that the permeability of the membrane was not degraded by ultrasound in the ranges of acoustic pressure and irradiation time in this study.

Epiphytic bacteria antagonistic to Curvularia leaf spot of yam

Curvularia eragrostidis yam leaf spot is a serious concern among the northeast Brazilian yam growing areas. In order to study its biocontrol, bacterial isolates from the yam phylloplane were tested against the pathogen. They were evaluated with respect to the following parameters: (1) inhibition of C. eragrostidis mycelial growth by using paired culture and cellophane membrane methods, (2) inhibition of conidium germination by using a paired suspension test, (3) reduction of disease severity and, (4) persistence of antagonistic action, on plants under greenhouse conditions. From a total of 162 bacterial isolates, 39 showed antagonism to the pathogen in paired culture. The bacteria produced extracellular, nonvolatile, and diffusible metabolites in the membrane cellophane test. Seventeen isolates resulted in more than 75% inhibition of C. eragrostidis mycelial growth. Among them, IF-26 showed the greatest antagonism. The isolates IF-82, IF-88, and IF-109 inhibited pathogen conidial germination, with average inhibition levels of 99.2, 98.2 and 96.2%, respectively. Under greenhouse conditions the antagonists were applied at three different time intervals relative to C. eragrostidis inoculation: 3 days before, at the same time, and 3 days after. IF-82 and IF-88 applied at the same time as pathogen inoculation both reduced disease severity 75%. IF-82 showed the best persistence of antagonistic action, with an average of 96.3%. IF-82, identified as Bacillus subtilis, was the best biocontrol agent for the yam leaf spot disease in this study.