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PubMed1998

A cluster of charged and aromatic residues in the C-terminal portion of maltoporin participates in sugar binding and uptake.

The maltoporin LamB of Escherichia coli K12 is a trimeric protein which facilitates the diffusion of maltose and maltodextrins through the bacterial outer membrane, and also acts as a non-specific porin for small hydrophilic molecules as well as a receptor for phages. Loop L9 (residues 375 to 405) is the most distal and largest surface-exposed loop of LamB. It comprises a central portion, which varies in size and sequence in the maltoporins of known sequence, flanked by two conserved regions containing charged and aromatic residues. In order to identify the residues within the proximal region that are specifically involved in sugar utilization, we used site-directed mutagenesis to change, individually, each of the charged (five) and aromatic (three) residues in the region 371 to 379 into alanine. None of the eight single amino acid substitutions affected the phage receptor activity of LamB. In contrast, they all affected, to variable extents, maltoporin functions. For all the mutants, very good correlations were observed between the effects on sugar binding and on in vivo uptake. In no case were maltoporin functions completely abolished. Mutants E374 A and W376 A were the most impaired (with over 60% reduction in dextrin binding and in vivo uptake of maltose and maltopentaose). These two mutations also led to an increased bacterial sensitivity to bacitracin and vancomycin. The functional and structural implications are discussed.

Amino Acid Sequence↗

PubMed2006

Stability and release of antiviral drugs from ethylene vinyl acetate (EVA) copolymer.

The use of polymer based drug delivery systems in dentistry is a relatively new area of research with the exception of the inhibition of secondary caries by the release of fluoride ions from polyalkenoate cements and their predecessors silicate cements. The present study was to test on orally biocompatible material, ethylene vinyl acetate copolymer (EVA), for release of antiviral drugs at oral therapeutic levels over extended periods of time. We also determined their stability during film casting and release. Materials studied include gancyclovir (GCY), acyclovir (ACY), dichloromethane (DCM), and ethylene vinyl acetate (EVA). The square films (3 x 3 x 0.1 cm) were prepared from the dry sheet obtained by solvent evaporation of polymer casting solutions. These solutions were made of EVA and the drug (40:1) in 70 ml of dichloromethane at 38 degrees C. Then drug release characteristics from the drug loaded films were examined at 37 degrees C for a minimum of 14 days in 10 ml medium (ddwater) replaced daily. Kinetics of drug release were followed by spectral measurements using previously determined lambda(max) values (GCY = 250 nm; ACY = 253 nm). A minimum of three samples was tested and reproducible results were obtained. Drug stability (ACY) during film casting and its release was determined using 1H NMR spectrometer (Bruker DRX-500 and 400). Rate of drug release was determined from the part of the curve (rate vs. time) after the onset of the "burst." Although GCY has a larger molecular weight (255) than ACY (225), GCY exhibited about three times higher rate of release than ACY. This difference in rate values may be explained due to its relatively greater solubility in EVA, facilitating faster diffusion of the molecules through the channels present in EVA. This is consistent with the observation that the rate at which drug molecules diffuse through the channels of the polymer, can be increased by decreasing the molecular weight. In the case of ACY, the molecules may be undergoing molecular associations, perhaps dimerization or trimerization in addition to its lower solubility in EVA. The diffusion of ACY tends to be slower under these circumstances compared to GCY resulting in lower rate value than in the case of GCY. Biological studies revealed that ACY exhibited a remarkable decrease in a number of viral organisms present in virus infected cell culture system using real-time polymerase chain reaction (RT-PCR). NMR analysis indicates that the chemical structure of the drug remains stable during film casting process and release.

Acyclovir↗

PubMed1991

Ceratitis capitata brain adenylate cyclase and its membrane environment.

Adenylate cyclase activation by GTP and octopamine as well as basal activity (in the presence of Mg2+) have been studied as a function of membrane structure in plasma membranes from brain of the dipterous Ceratitis capitata. Benzyl alcohol and lidocaine, but not phenobarbital, inhibited the three activities to the same extent. Triton X-100-solubilized adenylate cyclase was also inhibited by benzyl alcohol and lidocaine, but not by phenobarbital. Results could be explained by an effect on the catalytic unit lipid environment, which would be maintained after solubilization, counteracting the effect of these drugs to facilitate lateral diffusion and coupling of adenylate cyclase components in the lipid bilayer. The observation that the insect adenylate cyclase is relatively insensitive to changes in bulk bilayer fluidity is strengthened by the absence of effect of phenobarbital on enzyme activities. Indeed, this compound was as active as lidocaine or benzyl alcohol in increasing bulk membrane fluidity. The response of C. capitata adenylate cyclase to changes in membrane fluidity is different from that recorded in mammalian systems. This may be functionally important and result from the fact that insects are not warm-blooded.

Adenylyl Cyclases↗

PubMed1977

Ca2+ transport mediated by a synthetic neutral Ca2+ -ionophore in biological membranes.

The effect of a synthetic neutral ligand on the Ca2+ permeability of several biological membranes has been investigated. The ligand had been previously shown to possess Ca2+ -ionophoric activities in artificial phospholipid membranes. The neutral ionophore is able to transport Ca2+ across the membranes of erythrocytes and sarcoplasmic reticulum, when lipophilic anions such as tetraphenylborate and carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) are present, presumably to facilitate the diffusion of the charged Ca2+ -ionophore complex across the hydrophobic core of the membrane. In mitochondria, the neutral ionophore promotes the active transport of Ca2+ in response to the negative membrane potential generated by respiration, in the presence of the specific inhibitor of the natural carrier ruthenium red.

Animals↗

PubMed1994

Influence of dye and protein location on photosensitization of the plasma membrane.

Two membrane-photosensitizing dyes were used to investigate whether selected sites in the plasma membrane vary in their sensitivity to damage by singlet oxygen (1O2*) and, if so, what factors are responsible for the variation. The relative ability of Rose bengal (RB) and merocyanine 540 (MC540), both of which localize in the plasma membrane and produce 1O2*, to photosensitize five plasma membrane functions in P388D1 cells was evaluated. The five membrane functions assessed were: plasma membrane potential, proline transport, facilitated glucose diffusion, 5'-nucleotidase activity, and dye exclusion. Photosensitization efficiency by RB varied by a factor of 188 for these membrane functions, whereas for MC540 a range of only 24 was found. RB was a more efficient photosensitizer than MC540 but the relative efficiencies varied with the membrane function. The wide range of P50 values for RB suggests that it binds selectively to membrane sites where it causes damage with high efficiency; possibly a non-1O2* mechanism is involved. In contrast, MC540 photosensitized the three membrane functions involving integral membrane proteins about equally suggesting that differences are due to small variations in the distribution of MC540 in the plasma membrane and/or variations in the inherent reactivity of the membrane targets with 1O2*. The results indicate that the lability of membrane sites to photosensitization depends both on their inherent reactivity with 1O2* and the relative location of specific protein and dye molecules.

5'-Nucleotidase↗

PubMed1988

Cationic environment and secretin secretion in canine duodenal mucosa in vitro.

We examined the effects of the divalent cation calcium (Ca2+) and the monovalent cations potassium (K+) and sodium (Na+) and different modalities that affect the fluxes of these cations on immunoreactive secretin (IRS) secretion from canine duodenal mucosa in vitro. In the absence of extracellular Ca2+, the basal IRS secretion was inhibited by 29%. Increased uptake of Ca2+ by passive diffusion and facilitated uptake of Ca2+ by ionophore A23187 had no effect on basal IRS secretion. However, Ca2+ channel blocking agents, LaCl3 and verapamil, inhibited basal IRS secretion by 18 and 33% respectively. High concentrations of extracellular K+ caused a dose-dependent but delayed stimulation of IRS secretion. K+-stimulated IRS secretion was not neurally mediated. Similarly, 9-aminoacridine, which blocks K+ exchange, mimicked the effects of K+. Valinomycin (10(-6) M) inhibited both K+-stimulated and 9-aminoacridine-stimulated IRS secretion. In the absence of extracellular Na+, there was a delayed inhibition of both basal and K+-stimulated IRS secretion. These results suggest that changes in cationic environment are associated with alterations in the secretion of IRS. High extracellular K+ concentration is conducive, and the absence of extracellular Na+ and Ca2+ is inhibitory to IRS secretion.

Aminacrine↗

PubMed1995

Analysis of anticholesterol antibodies using hydrophobic membranes.

An analytical immunoblotting procedure and a serological enzyme-linked immunosorbent assay (ELISA) for the characterization of antibodies to cholesterol are described. Hydrophobic membranes consisting of polyvinylidene fluoride (PVDF) are used to immobilize cholesterol for immunodetection by anti-sterol antibodies. To determine whether antibodies to cholesterol were induced after immunization with liposomal cholesterol, we separated total lipid extracts of very-low density lipoproteins by thin layer chromatography (TLC) on silica gel plates and transferred the separated lipid classes to PVDF membranes using isopropanol to facilitate passive diffusion. Lipid transfer was confirmed by exposure of membranes to iodine vapors or by staining of cholesterol with filipin complex. Serum from immunized mice reacted with cholesterol, whereas pre-immune serum or serum from mice injected with control liposomes did not bind. To determine the amount of anti-cholesterol activity in serum, we coated microtiter plates consisting of PVDF membrane wells with cholesterol. The PVDF membrane-based ELISA was found to be more reproducible and four-fold more sensitive than the conventional ELISA on polystyrene plates. These techniques may be useful in the analysis of anti-sterol antibodies and antibodies to other hydrophobic antigens.

Antibodies↗

PubMed1990

Crystallization and preliminary X-ray characterization of maltoporin from Escherichia coli.

Crystals of maltoporin (the bacteriophage lambda receptor of Escherichia coli) that diffract X-rays to 3 A resolution can be grown reproducibly. Maltoporin is an integral membrane protein, which forms a channel in the E. coli outer membrane that specifically facilitates the diffusion of maltose and maltodextrins. The crystals have a rhombic prismatic habit and belong to the orthorhombic space group C222(1) with unit cell dimensions a = 130 A, b = 213 A and c = 216 A. X-ray structure determination is underway.

Bacterial Outer Membrane Proteins↗

PubMed1991

Crystallization and preliminary X-ray analysis of phosphoporin from the outer membrane of Escherichia coli.

Phosphoporin is a pore-forming transmembrane protein that spans the outer membrane of Escherichia coli and facilitates the diffusion of phosphates and phosphorylated compounds. Phosphoporin has been crystallized in several different crystal forms, although only one appears to be suitable for X-ray analysis. These crystals, which are hexagonal plates, diffract X-rays to 3 A resolution and belong to the space-group P6(3)22, with unit cell dimensions a = b = 121 A and c = 111 A.

Bacterial Outer Membrane Proteins↗

PubMed1983

Skeletal muscle function in hypoxia: effect of alteration of intracellular myoglobin.

Enhancement of oxygen flux through myoglobin containing solutions due to myoglobin facilitated oxygen diffusion is well recognized and is most apparent under conditions of hypoxia. To examine the function of intracellular myoglobin in vivo under conditions of hypoxia, the in situ dog gastrocnemius-plantaris muscle was studied. Administration of 10% oxygen resulted in arterial PO2 values between 32 and 36 mm Hg. Muscle oxygen consumption and isometric tension generation were determined during sustained 3-Hz contractions before and after administration of hydrogen peroxide that converted functional myoglobin to forms incapable of reversible combination with oxygen. Muscle blood flow, perfusion pressure, and muscle oxygen delivery were unchanged by such treatment. Hydrogen peroxide administration resulted in oxidation of intracellular myoglobin with a resultant decrease of 37% in muscle oxygen consumption and 42% in tension generation after 20 min of stimulation. In a control group not receiving hydrogen peroxide muscle oxygen consumption and tension generation decreased by 13% and 12%, respectively. We conclude that intact functional myoglobin is important in maintaining muscle function in isometric exercise under conditions of hypoxia.

Animals↗

PubMed1994

Magnetically responsive photosensitizing reagents for possible use in photoradiation therapy.

The ability of a magnetically responsive material to function as a carrier for photosensitizing agents for use in photoradiation therapy (PRT) has been examined in vitro. The photosensitizer has been attached to the magnetically responsive matrix (Dynabeads) by non-specific adsorption, Intralipid-mediated adsorption and poly-L-lysine mediated adsorption. In these studies, it has been demonstrated that conditions of attachment of photosensitizer to the matrix may be adapted in order to facilitate a diffuse or highly localized photo-toxic effect on target cells in vitro. The authors believe that this system may represent a novel approach to targeting photosensitizing agents to specific areas, thereby circumventing some of the problems associated with conventional photoradiation therapy (PRT), particularly in hollow organs.

HeLa Cells↗

PubMed1996

Specific interaction of the protein-D2 porin of Pseudomonas aeruginosa with antibiotics.

The protein-D2 porin of Pseudomonas aeruginosa is lacking in carbapenem or fluoroquinolone-resistant strains and hence was thought to facilitate the diffusion of these antibiotics. We examined the effect of several antibiotics on the single channel conductivity of protein-D2 in planar lipid bilayers and found that fluoroquinolones and carbapenems at concentrations of around 1 mM caused closure of the protein-D2 channel. Tetracycline, ampicillin, piperacillin, and latamoxef did not exert any detectable effect on the protein-D2 channel activity.

Anti-Bacterial Agents↗

PubMed1994

Gas chromatography applied to the lactulose-mannitol intestinal permeability test.

Intestinal permeability can be modified by various illnesses, trauma and sepsis. Alterations of the intestinal wall can facilitate the diffusion of potentially harmful substances such as endotoxins, as well as bacterial translocation. We describe the validation of a capillary gas chromatographic method for the determination of mannitol and lactulose, used as intestinal permeability probes. The method is linear up to 3 g/l for mannitol and 300 mg/l for lactulose; recovery from overload samples is between 92 to 110%. Intra-assay coefficients of variation (C.V.s) were 2.7 and 6.8% for mannitol and lactulose, respectively, and inter-assay C.V.s were 8.9 and 9.3%. Normal values for 25 healthy subjects (mean +/- S.D.) were 14.5 +/- 3.1% and 0.27 +/- 0.15% for mannitol and lactulose, respectively. The GC method presented is rapid and precise.

Adult↗

PubMed1991

The relationship between skin maturation and electrical skin impedance.

When performing electrophysiological testing, high electrical impedance values are sometimes found in neonates. Since excessive impedance can invalidate test results, a study was conducted to delineate the relationship between skin maturation and electrical skin impedance. This study investigated the skin impedance in 72 infants ranging from 196 to 640 days of age from conception. Regression analyses demonstrated a significant relationship between impedance and age, with the highest impedance centered around full-term gestation with values falling precipitously at time points on either side. Clinically, impedance values fall to normal levels at approximately four months following full-term gestation. Skin impedance values are low in premature infants, but rapidly increase as the age approaches that of full-term neonates. Low impedance values in premature infants are attributed to greater skin hydration which results from immature skin conditions such as 1) thinner epidermal layers particularly at the transitional and cornified layers; 2) more blood flow to the skin; and 3) higher percentage of water composition. These factors facilitate the diffusion of water vapor through the skin. As the physical barrier to skin water loss matures with gestational age, the skin impedance reaches a maximum value at full term neonatal age. After this peak, a statistically significant inverse relationship exists between electrical skin impedance and age in the first year of life. This drop in skin impedance is attributed to an increase in skin hydration as a result of the greater functional maturity of eccrine sweat glands.

Age Factors↗

PubMed1995

The influence of BIBW22BS, a dipyridamole derivative, on the antiproliferative effects of 5-fluorouracil, methotrexate and gemcitabine in vitro and in human tumour xenografts.

Dipyridamole is known as a potent inhibitor of facilitated diffusion-mediated nucleoside transport as well as a modulator of 'classical' multidrug resistance. BIBW22BS, a derivative of dipyridamole, has been found to be 20- to 100-fold more potent in the reversal of multidrug resistance when compared to the parent compound. In parallel, we studied the efficacy of BIBW22BS in the modulation of the antiproliferative effects of 5-fluorouracil, methotrexate and gemcitabine in human cancer cell lines. BIBW22BS, at non-toxic concentrations up to 1.0 microM, increased the antiproliferative effects of 5-fluorouracil 2- to 6-fold in seven of the eight colon cancer cell lines tested in a dose-dependent manner. The addition of 1.0 microM BIBW22BS to methotrexate resulted in a slight increase in the antiproliferative effects, but inhibited the activity of gemcitabine 30- to 100-fold in various cancer cell lines. In vitro, no notable difference was found between BIBW22BS and dipyridamole in their capacity to modulate the activity of the antimetabolites studied. BIBW22BS did not affect the growth inhibition induced by 5-fluorouracil or gemcitabine in human tumour xenografts grown subcutaneously in nude mice. We confirmed the higher potency of BIBW22BS when compared to dipyridamole in the reversal of drug resistance in the Pgp-positive COLO 320 cell line.

Animals↗

PubMed2005

Cd2+ transport and storage in the chloroplast of Euglena gracilis.

Euglena gracilis lacks a plant-like vacuole and, when grown in Cd2+-containing medium, 60% of the accumulated Cd2+ is located inside the chloroplast. Hence, the biochemical mechanisms involved in Cd2+ accumulation in chloroplast were examined. Percoll-purified chloroplasts showed a temperature-sensitive uptake of the free 109Cd2+ ion. Kinetics of the uptake initial rate was resolved in two components, one hyperbolic and saturable (Vmax 11 nmol 109Cd2+ min(-1) mg protein (-1), Km 13 microM) and the other, linear and non-saturable. 109Cd2+ uptake was not affected by metabolic inhibitors or illumination. Zn2+ competitively inhibited 109Cd2+ uptake (Ki 8.2 microM); internal Cd2+ slightly inhibited 109Cd2+ uptake. Cadmium was partially and rapidly released from chloroplasts. These data suggested the involvement of a cation diffusion facilitator-like protein. Chloroplasts isolated from cells grown with 50 microM CdCl2 (ZCd50 chloroplasts) showed a 1.6 times increase in the uptake Vmax, whereas the Km and the non-saturable component did not change. In addition, Cd2+ retention in chloroplasts correlated with the amount of internal sulfur compounds. ZCd50 chloroplasts, which contained 4.4 times more thiol-compounds and sulfide than control chloroplasts, retained six times more Cd2+. The Cd2+ storage-inactivation mechanism was specific for Cd2+, since Zn2+ and Fe3+ were not preferentially accumulated into chloroplasts.

Animals↗

PubMed2006

Cell membrane damage induced by lacticin 3147 enhances aldehyde formation in Lactococcus lactis IFPL730.

Amino acid catabolism is mainly initiated in Lactococcus lactis by a transamination reaction that leads to the formation of alpha-keto acids. In addition, a novel alpha-keto acid decarboxylase enzyme, rare in lactic acid bacteria, responsible for the conversion of alpha-keto acids into aldehydes has been reported in L. lactis IFPL730. The effect of lacticin 3147-induced cell damage on both amino acid transamination and alpha-keto acid decarboxylation by L. lactis IFPL730 leading to the formation of aldehydes from amino acids was investigated. Cell membrane permeabilization induced by lacticin 3147 facilitated the diffusion of amino acids into the cells and thus, enhanced amino acid transamination and formation of alpha-keto acids. However, alpha-keto acid decarboxylation was not affected by cell membrane permeabilization since decarboxylation of alpha-keto acids in both control and lacticin 3147-treated cells were similar, suggesting that these substrates could freely diffuse inside the cells. Nevertheless, the formation of 2-methylbutyraldehyde from isoleucine was enhanced in lacticin 3147-treated cells. The increase in alpha-keto acids formation rate by L. lactis IFPL730 due to lacticin 3147-induced cell damage, led to a concomitant increase in the subsequent decarboxylation reaction that complete the metabolic pathway to aldehyde production from amino acids. The present study points out to the use of the food grade lacticin 3147 along with L. lactis IFPL730 as a valuable tool in the development of cheese flavour.

Aldehydes↗

PubMed2006

Ni/SiO2 promoted growth of carbon nanofibers from chlorobenzene: characterization of the active metal sites.

The temporal changes to supported Ni sites during the growth of graphitic carbon nanofibers (GCNs) via the decomposition of chlorobenzene over Ni/SiO2 at 873 K have been investigated. The reaction of chlorobenzene with hydrogen also generated benzene, via catalytic hydrodechlorination, as the principal competing reaction. Reaction selectivity was found to be time dependent with a switch from a preferential hydrodechlorination to a predominant decomposition that generated an increasingly more structured carbon product over prolonged time-on-stream. These findings are discussed in terms of Cl/catalyst interaction(s) leading to metal site restructuring, the latter manifest in a sintering and faceting of the Ni metal particles. The pressure exerted on the metal/support interface due to fiber formation was of sufficient magnitude to extract the Ni particle from the support; the occurrence of an entrapped Ni particle at the fiber tip is a feature common to the majority of GCNs with the incorporation of Ni fragments along the length of the GCN. Metal site restructuring has been probed by temperature-programmed reduction of the passivated samples, H2 chemisorption/temperature-programmed desorption (TPD) and XANES/EXAFS analyses. This restructuring serves to enhance destructive chemisorption and/or facilitate carbon diffusion to generate the resultant GCN. The nature of the carbonaceous product has been characterized by a combination of TEM-EDX, SEM, XRD and temperature-programmed oxidation (TPO).

Adsorption↗