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PubMed1988

Three-dimensional structure of the tryptophan synthase alpha 2 beta 2 multienzyme complex from Salmonella typhimurium.

The three-dimensional structure of the alpha 2 beta 2 complex of tryptophan synthase from Salmonella typhimurium has been determined by x-ray crystallography at 2.5 A resolution. The four polypeptide chains are arranged nearly linearly in an alpha beta beta alpha order forming a complex 150 A long. The overall polypeptide fold of the smaller alpha subunit, which cleaves indole glycerol phosphate, is that of an 8-fold alpha/beta barrel. The alpha subunit active site has been located by difference Fourier analysis of the binding of indole propanol phosphate, a competitive inhibitor of the alpha subunit and a close structural analog of the natural substrate. The larger pyridoxal phosphate-dependent beta subunit contains two domains of nearly equal size, folded into similar helix/sheet/helix structures. The binding site for the coenzyme pyridoxal phosphate lies deep within the interface between the two beta subunit domains. The active sites of neighboring alpha and beta subunits are separated by a distance of about 25 A. A tunnel with a diameter matching that of the intermediate substrate indole connects these active sites. The tunnel is believed to facilitate the diffusion of indole from its point of production in the alpha subunit active site to the site of tryptophan synthesis in the beta active site and thereby prevent its escape to the solvent during catalysis.

Amino Acid Sequence↗
PubMed1979

Biology of Toxoplasma gondii, its survival in body tissues and liquids, risks for the pregnant woman.

After briefly outlining the biology of Toxoplasma gondii the authors discuss the resistance of the parasite in body liquids and tissues under certain environmental conditions. Parasite resistance and its consequent risk of human infection, particularly for the pregnant woman, is emphasized. Toxoplasma oocysts, the sexual expression of the parasite, and the cyst facilitate the diffusion of Toxoplasma, as they are much more resistant than the trophozoite.

Animals↗
PubMed1981

[Calcium desorption from the cornea while using complex-forming substances].

The time course of calcium desorption from the eye cornea and the efficacy of the use of complex-forming substances during desorption were examined. Experiments with labeled calcium showed an active absorption of the ions by the normal cornea thereby making it turbid. The removal of labeled calcium from an isolated eye and from corneal pieces while using a combination of complex-forming substances and diffusion facilitating-papain was accompanied by the lowering of tissue turbidity. Application of such combinations might eliminate, in some cases, the necessity of surgical operations on the cornea.

Absorption↗
PubMed1982

[Interaction of NADH-dehydrogenase from M. lysodeikticus membranes with lipids in a reconstituted system].

The NADH-dehydrogenase isolated from the M. lysodeikticus membranes was reconstituted into liposomes from the lipids obtained from the same membranes. The presence and degree of the reconstitution were investigated by two-dimensional immunoelectrophoresis and photoreactive hydrophobic label. The quenching of protein fluorescence by the aqueous quencher J- was practically the same for the enzyme in the reconstituted system and in the detergent solution, whereas the quencher interacting with the membrane--cetylpyridinium chloride--was effective in the first case and not effective in the second one. Evidence for the energy transfer from protein chromophores of NADH dehydrogenase in the proteoliposomes (lambda excit = 286 nm) to the hydrophobic fluorescent probe pyrene was obtained. It was found that about 30% of the chromophores in the enzyme molecule are involved in this process. The hydrophobic spin probe, whose paramagnetic fragment is located on the surface and not inside the hydrophobic phase of the membrane, can act as electron acceptor during NADH oxidation in the reconstituted system. The data obtained are suggestive of the exposure of the bulk of the enzyme molecule to the environment and of interaction of the smaller part of the molecule with the lipid phase. The active center is located on the part of the enzyme molecule which is exposed to water. It is assumed that the NADH-dehydrogenase molecule is exposed to water. It is assumed that the NADH-dehydrogenase molecule is involved in heat diffusion which facilitates the active center interaction with the membrane surface.

Cell Membrane↗
PubMed1982

Use of rapid superfusion to differentiate the release of dopamine from striatal tissue induced by sympathomimetic amines from release induced by potassium.

A rapid superfusion system incorporating a continuous amperometric detector has been employed to study the effects of p-tyramine, d-amphetamine and cocaine on K+-stimulated release of dopamine (DA) from striatal tissue. Qualitative identification of the released substances is made by liquid chromatography with electrochemical detection and DA is established to be the principal substance released following exposure of the tissue to 60 mM K+ in the presence of Ca++. At a superfusion rate of 1 ml/min with a 20 microliter volume tissue holder, d-amphetamine and p-tyramine do not elicit detectable release of endogenous substances. These results are in direct contrast to the significant release of DA observed when striatal tissue is incubated with d-amphetamine for the same time period and suggest that the pools susceptible to release by the sympathomimetic amines are removed by superfusion. When striatal tissue is superfused with a buffer containing 60 mM K+ and cocaine or sympathomimetic amines, the effects of uptake blockade are manifested as an increase in the amount and duration of observed release over that obtained with 60 mM K+. In a competitive study of cocaine and p-tyramine with d-amphetamine, it appears that all three of these agents induce blockade at the same site(s). All of these data are in direct accord with the facilitated exchange diffusion mechanism proposed for the actions of sympathomimetic amines at DA nerve terminals.

Animals↗
PubMed1995

Novel aspects of the ultrastructural organization of human corneal keratocytes.

PURPOSE: Proper functioning of the endothelium and proper structural organization of the keratocytes and collagen bundles are of ultimate importance for transparency of the cornea. The role of the endothelium has been investigated extensively, whereas the role of the keratocytes is still unclear. Detailed knowledge on the ultrastructural organization of keratocytes and the relationship between keratocytes and collagen bundles is as essential for understanding corneal transparency as is knowledge of endothelial functioning. METHODS: Thirty-five corneas (30 postmortem donor corneas and 5 fresh corneas from the operating theater; age range, 28 to 90 years) were used for light microscopy, transmission electron microscopy, and scanning electron microscopy. Serial frontal sections of the central stroma reaching from epithelium to endothelium and cross-sections were studied. At three levels, reconstructions of the mutual arrangement of keratocytes were made using semithin sections. RESULTS: Keratocytes have the appearance of highly active cells with an abundancy of organelles. Between the dendritic ramifications of these cells, large amounts of amorphous material is observed. One of the most remarkable observations is the presence of an extensive network of fenestrations along the surface of the keratocytes. Another important observation is the circular arrangement of keratocytes gradually turning clockwise like a corkscrew from epithelium to endothelium. CONCLUSIONS: From the current study, the following conclusions can be drawn: Keratocytes are not quiescent but are highly active cells probably involved in turnover of the extracellular matrix; fenestrations may be of functional relevance with respect to facilitation of diffusion and mechanical attachment of the collagen fibers to the keratocytes; the corkscrew organization of keratocytes suggests that they form completely closed sheets of communicating cells throughout the depth of the cornea, creating equal chances for all light rays to pass one or more keratocytes and thus minimizing variation in light scattering over the entire cornea.

Adult↗
PubMedDate not supplied

Influence of cell surface hydrophobicity on attachment of Candida albicans to extracellular matrix proteins.

Cell surface hydrophobicity expression by Candida albicans facilitates a diffuse binding distribution of yeast cells to host tissues ex vivo. One possibility for the receptor site responsible for the binding pattern of hydrophobic cells is the extracellular matrix (ECM). In this study, we evaluated the interaction of hydrophobic and hydrophilic C. albicans with ECM proteins immobilized onto wells of microtitre tissue. Culture plates, and the ability of ECM proteins to block the binding of hydrophobic cells to splenic tissue ex vivo. Hydrophobic C. albicans bound in greater numbers than hydrophilic cells to the immobilized proteins, particularly fibrinogen, fibronectin, collagen type IV and laminin. Similar results were obtained regardless of C. albicans strains or of growth medium. Collagen and fibronectin blocked the binding of hydrophobic cells to the white pulp but not to the marginal zones in splenic tissues when tested with the ex vivo assay. These results suggests that the diffuse binding pattern of hydrophobic cells in the ex vivo assay may be due to their enhanced ability over hydrophilic cells to bind to ECM proteins, particularly fibronectin and collagen type IV.

Animals↗
PubMed1996

Role of the Golgi apparatus in the phosphorylation of apolipoprotein B.

Rat hepatic Golgi apparatus-rich fractions were utilized in an in vitro phosphorylation system containing [gamma-32P]ATP to investigate the phosphorylation of apolipoproteins (apo) B48 and B100. Our results demonstrate that the Golgi apparatus contains a kinase(s) that phosphorylates both apoB48 and apoB100 as well as 290- and 460-kDa proteins recognized by antibody to apoB. We refer to the latter proteins as apoB57 and apoB90, respectively. Phosphorylations in the presence of Triton X-100, which increases the permeability of the membranes, or alamethicin, an ionophore that facilitates transmembrane diffusion of ATP, indicate that the active site of the kinase is on the luminal side of the membranes. However, studies with EDTA and EGTA, which are inhibitory to the kinase, suggest binding sites for Mg2+ and perhaps Ca2+ on the cytosolic membrane face. Phosphorylation of apoB was not stimulated by cAMP nor inhibited by protein kinase inhibitor peptide (5-24), indicating that cAMP dependent protein kinase was not involved in the phosphorylation process. Sodium carbonate treatment of the phosphorylated fraction, which permits separation of membrane and luminal contents, revealed that phosphorylated apoB90 and apoB57 are associated primarily with the membrane, whereas phosphorylated apoB48 is found in luminal contents as well as with the membranes. Phosphorylated apoB100 was found primarily with the membrane fraction. No evidence was found for phosphorylation of apoB in rough endoplasmic reticulum fractions. These studies demonstrate the importance of the Golgi apparatus as a subcellular site for the phosphorylation of apoB and suggest that apoB phosphorylation may be important in the assembly and secretion of apoB-containing lipoproteins.

Adenosine Triphosphate↗
PubMed1997

[Continuous intrathecal injection of baclofen in generalized tetanus. A therapeutic alternative].

BACKGROUND: In France, tetanus is diagnosed predominantly in the elderly due to the absence of vaccination in this population. Mortality remains high and is mainly related to complications secondary to prolonged hospitalization, intravenous administration of benzodiazepines, and at times curares. Baclofen, a selective agonist of GABAB receptors, directly counteracts the effects of the tetanic toxin. CASE REPORT: We report the case of a 92-year-old man suffering from tetanus, who fully recovered after a myorelaxant treatment based solely on intrathecal baclofen administration. Continuous intrathecal administration of baclofen resulted in a marked muscular relaxation with minor side effects (bradycardia, sleepiness), which rapidly stopped after the cessation of the treatment. Initial careful titration is required in order to determine the optimal dose of baclofen which provides a substantial myorelaxation together with minor central effects. Low concentration of baclofen may facilitate its diffusion. DISCUSSION: Continuous intrathecal administration of barclofen appears to be a valuable alternative therapy in tetanus. This new therapeutic modality promises to improve the prognosis of this condition, especially in the elderly.

Aged↗
PubMed1998

[Anatomical description of a natural perforation present in the human lumbar pia mater].

OBJECTIVE: The pia mater has always been considered more permeable than other meningeal membranes. Natural pia mater perforations found in some animals at-test to this membrane's permeability. Such perforations, however, have never been demonstrated in human tissue. Our objective was to study human pia mater from the dorsal lumbar region, looking for perforations that facilitate the diffusion of substances to the spinal cord following subarachnoid administration. MATERIAL AND METHOD: The specimens were removed from four human cadavers aged 70, 72, 77 and 78 years between 8 and 12 hours after death. The specimens were fixed in a phosphate glutaraldehyde buffer solution, followed by desiccation in acetone and critical point elimination of acetone, treatment with carbon and metallization with gold. RESULTS: The pia mater was composed of a smooth surfaced, thin layer of cells and underlying connective tissue formed mainly of collagen fibers and fundamental amorphous matter. The collagen fibers were oriented in various directions. Throughout the surface of the pia mater, natural circular, elliptical and ovoid perforations were distributed irregularly. Size varied. Most measured over 10 to 15 micrometers in diameter or less than 5 to 8 micrometers. Inside the openings, fibers similar to collagen fibers could be seen at the point where they would normally be found beneath the cell layer. CONCLUSION: The total thickness of the pia mater varies in different zones of the spine, as a result of variations in the thickness of the cell layer and in the underlying extracellular layer. The existence of natural fenestrations in all the analyzed specimens of human dorsal lumbar pia mater accounts for the high permeability of this membrane, which permits substances administered in spinal fluid to reach the spinal cord. These natural fenestrations are areas where the cell surface in absent, with underlying collagen fibers usually visible. The pia mater is generally believed to be composed of a complete cell layer that forms a barrier between the central nervous system and the subarachnoid space; however, the presence of fenestrations would indicate that such a barrier does not exist, the base membrane being placed under the connective fibers, the only intact structure prior to medullary glial cells.

Aged↗
PubMed2005

Increased hepatocellular uptake of long chain fatty acids occurs by different mechanisms in fatty livers due to obesity or excess ethanol use, contributing to development of steatohepatitis in both settings.

Long chain fatty acids (LCFA) enter cells by both facilitated transport and diffusion, the former accounting for > or = 90%. Facilitated LCFA transport is up-regulated in adipocytes from obese rats, mice, and humans. To clarify the role of hepatocellular LCFA uptake in hepatic steatosis (fatty liver), [3H]-oleic acid (OA) uptake was studied in hepatocytes isolated from Zucker fatty(Z) and control(C) and ethanol-fed(E) Wistar rats, and demonstrated both saturable and non-saturable components, each a function of the unbound OA concentration ([OAu]). The uptake Vmax was identical in C and Z animals, but was increased 2.4-fold in E animals (p < 0.01). The non-saturable uptake rate constant did not differ between groups. Plasma LCFAs averaged 600 microM in C and E and 1,200 microM in Z animals. Total LCFA uptake averaged 1.35, 2.78 and 3.54 pmol/sec/50,000 cells in C, Z, and E animals, respectively. A 2-fold uptake increase in Z animals, in which Vmax was unaltered, entirely reflected the increased plasma LCFA concentration, whereas the 2.6-fold increase in E animals, in which plasma LCFA were not increased, resulted from up-regulation of facilitated transport. Thus, while increased hepatic LCFA uptake contributes to pathogenesis of hepatic steatosis in both obesity and excessive ethanol consumption, the mechanisms differ.

Animals↗
PubMed1990

Acidic mucin layer facilitates micelle dissociation and fatty acid diffusion.

The presence of a mucin layer on the surface of the intestinal epithelium has been suggested as an important factor in maintaining an acidic microclimate. The presence of such a low-pH compartment has been shown to facilitate fatty acid uptake. The mechanisms leading to the enhancement of fatty acid uptake were investigated in a purified acidic mucin layer. Our results indicate that the presence of a low-pH compartment indeed facilitates the dissociation of mixed micelles made of taurocholate and oleic acid. The released fatty acid formed an emulsion at the mucin layer, and this event could be visualized by the naked eye. When the size of the particles in the micelle solution was examined by photon correlation spectroscopy, it was found that acidification alone can lead to the formation of particles with size substantially greater than that of micelles. With the use of labeled fatty acid, the change in optical density can be correlated to the amount of fatty acid appearing in the mucin layer in an asymptotic fashion, suggesting that using the turbidity as an indicator might underestimate fatty acid diffusion. Despite this limitation, the rate of fatty acid diffusion in the mucin layer was estimated to be 400% of that in the buffer solution.

Animals↗
PubMed2002

Study on protein adsorption kinetics to a dye-ligand adsorbent by the pore diffusion model.

Adsorption kinetics of bovine serum albumin (BSA) and bovine hemoglobin (bHb) to Cibacron Blue 3GA (CB) modified Sepharose CL-6B has been studied. The effects of liquid-phase ionic strength and CB coupling density on the uptake rates of these two proteins in Tris-HCl buffer (pH 7.5) were evaluated by effective pore diffusivity derived from a pore diffusion model. The results showed that despite their similar molecular masses and sizes, the effects of aqueous-phase ionic strength and CB density on the effective pore diffusivities of BSA and bHb were distinctly different. The effective pore diffusivity of BSA to CB-Sepharose increased significantly with decreasing CB density and increasing liquid-phase ionic strength. This was considered due to the decrease in electrostatic repulsion between the BSA and CB molecules of like charge. That is, the increase in ionic strength and the decrease in CB coupling density reduced the electrostatic hindrance effect on BSA diffusion to CB-Sepharose, facilitating the hindered pore diffusion. In contrast, because of the higher isoelectric point of bHb (7.0) compared to BSA (4.7), bHb suffered little electrostatic hindrance effect during its diffusion to CB-Sepharose. Therefore, the effective pore diffusivity of bHb was unchanged with the change in liquid-phase ionic strength and CB coupling density.

Adsorption↗
PubMed1969

Solubilities and diffusivities of oxygen in hemolyzed human blood solutions.

By continuous absorption and by bubble collapse methods respectively, the solubilities and diffusion coefficients of oxygen in water and in dilute solutions of human hemoglobin (1.11, 2.22, and 4.44 wt%) have been determined at one atmosphere and 10 degrees , 20 degrees , 30 degrees , 40 degrees , 50 degrees , and 60 degrees C. Measured equilibrium constants, oxygen/hemoglobin ratios and isochoric heats of solution have been interpreted in terms of various mechanisms for oxygen-hemoglobin interaction. Oxygen diffusivities obtained experimentally for the hemolyzed blood solutions have been found to compare favorably with those predicted by a model of facilitated transport proposed by Houghton (1966). The diffusion measurements indicate that, while kinetic phenomena cannot be ignored, the over-all rate of exchange of oxygen with hemoglobin is not a controlling factor in facilitated diffusion. Anomalous equilibrium constants and temperature coefficients have been observed in the most dilute hemoglobin solution at the lowest temperatures.

Biological Transport↗
PubMed2003

Human embryonic germ cell derivatives facilitate motor recovery of rats with diffuse motor neuron injury.

We have investigated the potential of human pluripotent cells to restore function in rats paralyzed with a virus-induced motor neuronopathy. Cells derived from embryonic germ cells, termed embryoid body-derived (EBD) cells, introduced into the CSF were distributed extensively over the rostrocaudal length of the spinal cord and migrated into the spinal cord parenchyma in paralyzed, but not uninjured, animals. Some of the transplanted human cells expressed the neuroglial progenitor marker nestin, whereas others expressed immunohistochemical markers characteristic of astrocytes or mature neurons. Rare transplanted cells developed immunoreactivity to choline acetyltransferase (ChAT) and sent axons into the sciatic nerve as detected by retrograde labeling. Paralyzed animals transplanted with EBD cells partially recovered motor function 12 and 24 weeks after transplantation, whereas control animals remained paralyzed. Semi-quantitative analysis revealed that the efficiency of neuronal differentiation and extension of neurites could not account for the functional recovery. Rather, transplanted EBD cells protected host neurons from death and facilitated reafferentation of motor neuron cell bodies. In vitro, EBD cells secrete transforming growth factor-alpha (TGF-alpha) and brain-derived neurotrophic factor (BDNF). Neutralizing antibodies to TGF-alpha and to BDNF abrogated the ability of EBD-conditioned media to sustain motor neuron survival in culture, whereas neutralizing antibodies to BDNF eliminated the axonal outgrowth from spinal organotypics observed with direct coculture of EBD cells. We conclude that cells derived from human pluripotent stem cells have the capacity to restore neurologic function in animals with diffuse motor neuron disease via enhancement of host neuron survival and function.

Alphavirus Infections↗
PubMed1982

Facilitated transport of oxygen in the presence of membranes in the diffusion path.

Most of the experimental observations on facilitated transport have been done with millipore filters, and all the theoretical studies have assumed homogeneous spatial properties. In striated muscle there exist membranes that may impede the diffusion of the carrier myoglobin. In this paper a theoretical study is undertaken to analyze the transport in the presence of membranes in the diffusion path. For the numerical computations physiologically relevant values of the parameters were chosen. The numerical results indicate that the presence of membranes tends to decrease the facilitation. For the nonlinear chemical kinetics of the reaction of oxygen with the carrier, this decrement also depends on the location of the membranes. At the higher oxygen concentration side of each membrane the flow of combined oxygen is transferred to the flow of dissolved oxygen. The reverse process occurs at the lower concentration side. Jump discontinuities of the concentration of the oxygen-carrier compound at each membrane are associated with these transfers. The decrement of facilitation is due to the cumulative effect of these jump discontinuities.

Animals↗
PubMed1995

Fps1, a yeast member of the MIP family of channel proteins, is a facilitator for glycerol uptake and efflux and is inactive under osmotic stress.

The Saccharomyces cerevisiae FPS1 gene, which encodes a channel protein belonging to the MIP family, has been isolated previously as a multicopy suppressor of the growth defect of the fdp1 mutant (allelic to GGS1/TPS1) on fermentable sugars. Here we show that overexpression of FPS1 enhances glycerol production. Enhanced glycerol production caused by overexpression of GPD1 encoding glycerol-3-phosphate dehydrogenase also suppressed the growth defect of ggs1/tps1 delta mutants, suggesting a novel role for glycerol production in the control of glycolysis. The suppression of ggs1/tps1 delta mutants by GPD1 depends on the presence of Fps1. Mutants lacking Fps1 accumulate a greater part of the glycerol intracellularly, indicating that Fps1 is involved in glycerol efflux. Glycerol-uptake experiments showed that the permeability of the yeast plasma membrane for glycerol consists of an Fps1-independent component probably due to simple diffusion and of an Fps1-dependent component representing facilitated diffusion. The Escherichia coli glycerol facilitator expressed in a yeast fps1 delta mutant can restore the characteristics of glycerol uptake, production and distribution fully, but restores only partially growth of a ggs1/tps1 delta fps1 delta double mutant on glucose. Fps1 appears to be closed under hyperosmotic stress when survival depends on intracellular accumulation of glycerol and apparently opens rapidly when osmostress is lifted. The osmostress-induced High Osmolarity Glycerol (HOG) response pathway is not required for inactivation of Fps1. We conclude that Fps1 is a regulated yeast glycerol facilitator controlling glycerol production and cytosolic concentration, and might have additional functions.

Aquaporins↗