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PubMed1998

Cellular accumulation, localization, and activity of a synthetic cyclopeptamine in fungi.

A novel synthetic cyclopeptamine, A172013, rapidly accumulated by passive diffusion into Candida albicans CCH442. Drug influx could not be totally facilitated by the membrane-bound target, beta-(1,3)-glucan synthase, since accumulation was unsaturable at drug concentrations up to 10 microg/ml (about 1.6 x 10(-7) molecules/cell), or 25x MIC. About 55 and 23% of the cell-incorporated drug was associated with the cell wall and protoplasts, respectively. Isolated microsomes contained 95% of the protoplast-associated drug, which was fully active against glucan synthesis in vitro. Drug (0.1 microg/ml) accumulation was rapid and complete after 5 min in several fungi tested, including a lipopeptide/cyclopeptamine-resistant strain of C. albicans (LP3-1). The compound penetrated to comparable levels in both yeast and hyphal forms of C. albicans, and accumulation in Aspergillus niger was 20% that in C. albicans. These data indicated that drug-cell interactions were driven by the amphiphilic nature of the compound and that the cell wall served as a major drug reservoir.

Candida albicans↗
PubMed1994

A novel role for E- and P-selectins: shape control of endothelial cell monolayers.

The migration of neutrophils from blood vessels to peripheral tissues is a key step of inflammation. This requires the formation of transient gaps between endothelial cells with concomitant leucocyte squeezing through these narrow apertures and immediate restoration of endothelium continuity. It is currently considered that the main role of selectins is to mediate the initial contact between flowing leucocytes and endothelial cells. We show here that the binding of E- or P-selectins by specific antibodies induces a marked 'rounding up' of interleukin-1- or thrombin-activated human endothelial cells, respectively. Also, anti-E-selectin antibodies trigger a transient increase in cytosolic calcium involving intracellular calcium stores. No such effect is observed when von Willebrand factor or intercellular adhesion molecule 1 are similarly bound. Thus, in addition to promoting the initial interaction between activated endothelium and moving leucocytes, selectins might play a role in the induction of subsequent endothelial deformation, which would facilitate leucocyte arrest and transmigration towards peripheral tissues, and enhance the diffusion of soluble molecules between intravascular and peripheral compartments. Our results are consistent with this hypothesis and demonstrate a new property of endothelial selectins.

Antibodies, Monoclonal↗
PubMed2002

Neuronal calcium sensor-1 binds to regulated secretory organelles and functions in basal and stimulated exocytosis in PC12 cells.

Neuronal calcium sensor-1 (NCS-1) and its non-mammalian homologue, frequenin, have been implicated in a spectrum of cellular processes, including regulation of stimulated exocytosis of synaptic vesicles and secretory granules (SGs) in neurons and neuroendocrine cells and regulation of phosphatidylinositol 4-kinase beta activity in yeast. However, apart from these intriguing putative functions, NCS-1 and frequenin are relatively poorly understood. Here, the distribution, dynamics and function of NCS-1 were studied using PC12 cells that stably express NCS-1-EYFP (NCS-1 fused to enhanced yellow fluorescent protein) or that stably overexpress NCS-1. Fluorescence and electron microscopies show that NCS-1-EYFP is absent from SGs but is present on small clear organelles, some of which are just below the plasma membrane. Total internal reflection fluorescence microscopy shows that NCS-1-EYFP is associated with synaptic-like microvesicles (SLMVs) in growth cones. Overexpression studies show that NCS-1 enhances exocytosis of synaptotagmin-labeled regulated secretory organelles (RSOs) under basal conditions and during stimulation by UTP. Significantly, these studies implicate NCS-1 in the enhancement of both basal and stimulated phosphoinositide-dependent exocytosis of RSOs in PC12 cells, and they show that NCS-1 is distributed strategically to interact with putative targets on the plasma membrane and on SLMVs. These studies also reveal that SLMVs undergo both fast directed motion and highly hindered diffusive motion in growth cones, suggesting that cytoskeletal constituents can both facilitate and hinder SLMV motion. These results also reveal interesting similarities and differences between transport organelles in differentiated neuroendocrine cells and neurons.

Animals↗
PubMed1995

The collagen fibrils arrangement in the Wharton's jelly of full-term human umbilical cord.

In the present study, a cell maceration/scanning electron microscopy method is employed in order to reveal the three-dimensional organization of the collagen fibrils in the Wharton's jelly of the human umbilical cord at term. By this method the jelly appears as a three-dimensional network of collagen fibrils that runs continuously from the amniotic membrane to the umbilical vessels. A diverse architecture of the collagen fibrillar network in the inner and outer region of the cord is observed suggesting an anatomical as well as a functional compartmentalization. In fact, the network is soft in the inner part and it is characterized by canalicular structures whereas in the outer region the collagen is dense and the network is characteristically composed by a sponge-like system of cavernous spaces. It is suggested that these cavities might store the ground substance and allow its diffusion during twisting or compression of the cord. Furthermore they may facilitate the flow of metabolites throughout the jelly from and to the umbilical vessels and the amniotic cavity.

Amnion↗
PubMed1984

Evidence for a folded conformation of methionine- and leucine-enkephalin in a membrane environment.

Transfer of an aqueous-soluble peptide hormone or neurotransmitter such as [Met]- or [Leu]enkephalin (Tyr1-Gly2-Gly3-Phe4-Met5(Leu5)), to the lipid-rich environment of its membrane-embedded receptor protein may convert the peptide into a ("bioactive") conformation required for eliciting biological activity. We have examined by high-resolution nuclear magnetic resonance (NMR) spectroscopy the conformational parameters of free enkephalin in aqueous solution versus those of enkephalin bound to lysophosphatidylcholine micelles using two approaches: 1) exchange rates, line broadening, coupling constants, and chemical shift changes of enkephalin backbone peptide N-H protons were measured for free and membrane-bound peptide in H2O (360 MHz, pH 5.6, 20 degrees C). A selective upfield shift observed for the Met5(Leu5) N-H proton upon lipid binding was interpreted in terms of its incorporation into an intramolecular H-bond. 2) 13C chemical shift changes induced by the shift reagent praseodymium nitrate (Pr(NO3)3) were compared in the presence and absence of lipid micelles. Significant changes occurring in Gly2 carbon atoms in membrane-bound enkephalin suggested the relative proximity of this residue to the Pr3+ atom (bound to the Met5(Leu5) COOH-terminal carboxylate 4 residues away). These combined results, in conjunction with studies on the specific interactions of enkephalin substituents with the micelles (Deber, C. M., and Behnam, B. A., (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 61-65) suggest that enkephalin folds into an intramolecularly H-bonded beta-turn structure (with an H-bond between Gly2 C = O and Met5 NH) in the lipid environment. Such folding could facilitate the positioning of strategic residues in vivo as the hormone diffuses toward its receptor.

Dimethyl Sulfoxide↗
PubMed1999

Olfactory ensheathing cells promote neurite extension from embryonic olfactory receptor cells in vitro.

The role of ensheathing cells, a macroglial cell type with a unique presence in the olfactory system, in the outgrowth of olfactory receptor cell neurites was explored in vitro. Glial cell cultures harvested from both the olfactory bulb nerve layer and the hippocampus were established and immunocytochemically characterized. The expression of the p75 low-affinity nerve growth factor receptor by ensheathing cells was used to distinguish them from other macroglial subpopulations. Results indicated that ensheathing cell cultures were approximately 80% pure. Olfactory receptor cells were cocultured with ensheathing or hippocampal glial cells or were seeded on laminin or poly-L-lysine as controls. Olfactory receptor cells extended the longest primary neurites when cocultured with ensheathing cells. Neurite extension on hippocampal glia and laminin was less extensive than that observed on ensheathing cells but higher than that on poly-L-lysine. The neurite outgrowth-promoting effect of ensheathing cells was, at least in part, mediated by diffusible factors, because olfactory receptor cell neurite extension could also be facilitated when receptor cells were cultured in ensheathing cell-conditioned media. In contrast, cortical neurons extended neurites of equivalent lengths on ensheathing and hippocampal glia. The results suggest that ensheathing cells may release factors that support the continuous outgrowth of olfactory receptor cell axons and, therefore, the capacity of this pathway to recover from injury.

Animals↗
PubMed2003

Structural aspects of DNA repair: the role of restricted diffusion.

DNA repair and protection processes impose arduous demands upon cellular systems. The high-fidelity recombinational repair pathway entails a rapid genome-wide search for sequence homology. The efficiency of this transaction is intriguing in light of the uniquely adverse diffusion traits of the involved species. DNA protection in cells exposed to continuous stress or prolonged starvation is equally enigmatic, because the ability of such cells to deploy energy-dependent enzymatic repair processes is hampered as a result of progressive perturbation of the intracellular energy balance. DNA repair in radio-resistant bacteria, which involves accurate chromosome reconstruction from multiple fragments, is similarly associated with apparently insurmountable logistical obstacles. The studies reviewed here imply that the mechanisms deployed to overcome these intrinsic hurdles have a basic common denominator. In all these cases, condensed and ordered chromatin assemblies are formed, within which molecular diffusion is restricted and confined. Restricted diffusion thus appears as a general strategy that is exploited by nature to facilitate homologous search, to promote energy-independent DNA protection through physical DNA sequestration and attenuated accessibility to damaging agents, and to enable error-free repair of multiple double-strand DNA breaks.

Animals↗
PubMed2004

Effect of the refractive index mismatch on light propagation through diffusive layered media.

The effect of the refractive index mismatch on light propagation through diffusive layers has been investigated. The refractive index mismatch changes the balance of energy inside the medium determining a temporal and spatial redistribution of light. Light penetration through the medium is obstructed (facilitated) by a negative (positive) refractive index step variation. An analytical solution of the time-dependent diffusion equation that accounts for this effect has been obtained. The solution has been validated by comparisons with the results of Monte Carlo simulations. An excellent description of light propagation is given even for a high refractive index mismatch.

Animals↗
PubMed2000

Characterization of the diffuse mucosal associated lymphoid tissue of feline small intestine.

Characterization of the feline intestinal mucosal associated lymphoid tissue (MALT) will facilitate investigation of intestinal disease in the cat and promote the cat as an animal model for a range of human diseases which involve the intestinal lymphoid tissue. This includes inflammatory bowel disease, viral and non-viral associated intestinal lymphomas and immunodeficiency associated syndromes. Morphologic and phenotypic characterization of the normal small intestinal diffuse MALT in 22 SPF cats was performed using flow cytometry and cytology on isolated intestinal leukocytes from the intra-epithelial and lamina proprial compartments, as well as immunohistology on tissues from the feline duodenum, jejunum and ileum. The intra-epithelial compartment (IEC) was dominated by lymphocytes (>85%) which frequently contained intracytoplasmic granules. The most striking findings in the IEC were the elevated percentages of CD8 alpha+ lymphocytes (40%), presumed to express CD8 alpha alpha chains, and CD4-/CD8- (double negative) lymphocytes (44%), and the consistent presence of a minor subpopulation of CD3+/CD11d+ IELs (6%). Small percentages of CD4+ lymphocytes (10%) were observed such that the IEL CD4:CD8 ratio (0.25) was low. The LPC also contained a majority of T cells and few plasma cells. However, this compartment had reduced percentages of CD8 alpha+ lymphocytes (28%) and increased percentages of CD4+ lymphocytes (27%) relative to the IEC. However, the LPL CD4:CD8 ratio (1.0) remained low compared with the ratio in peripheral blood. In feline MALT, MHC class II expression was lower than in other peripheral lymphoid compartments. The results of this study provide important reference values for future investigations involving feline intestinal lymphocytes and demonstrates that the leukocyte distribution and phenotypic characteristics of the feline diffuse MALT appear largely similar to the murine, rat and human counterparts.

Animals↗
PubMed1983

Implementing asthma self-management education in medical care settings--issues and strategies.

Asthma self-management education has the potential to improve the health and quality of life for children and to reduce use of acute care services. The preparation of parents and children to prevent asthma attacks and to ameliorate acute episodes when they do occur can best be accomplished with the support of medical care providers. This analysis addresses policy issues relevant to that acceptance: the degree of integration into medical practice that is desirable; the most hospitable clinical settings; and the incentives for providers, consumers, and payers that would result in appropriate supply and use of ASME:. The analysis commences with a discussion of the costs and benefits of making ASME a part of medical care. It then turns to a description of the trends that led to the current array of institutions, services, and incentives within the health care sector. Future changes in family context, financing, and other determinants of the use of medical services by children and youth are forecast. The framework for the analysis of incentives is diffusion of innovation. ASME is analyzed according to the attributes that generally facilitate the acceptance of a new procedure. In its current form and with current incentives, ASME is fairly complex and difficult to communicate, has little relative advantage, and is not necessarily compatible with the usual provider goals. The motivating factors for three interrelated groups-providers, consumers, and third-party payers-and leverage points for the application of incentives are identified. The latter include reimbursement, standards of practice, information generation and flow, insurance regulation, institutional development, and research funding. Important information to potential adopters includes the definition of the service, its optimal intensity and mix of providers, and its effects on net costs and quality of life.

Asthma↗
PubMed2001

Low doses of N-methyl-D-aspartate antagonists in superficial laminae of medulla oblongata facilitate wind-up of convergent neurones.

In this study, a trigeminal model was used in which high threshold C-fibre-evoked activities of convergent neurones located in the subnucleus oralis of the trigeminal complex are modulated through the superficial part, the substantia gelatinosa, of the subnucleus caudalis. The two subnuclei are located 3 mm apart, therefore, it was possible to inject dizocilpine, a non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist, into either the superficial or the deep parts of subnucleus caudalis without interfering with ongoing recording of convergent neurones in subnucleus oralis. A differential NMDA-dependent modulation of wind-up was observed according to the dose and the injection target. (1) The injections of small non-diffusible doses (0.12 microg) of dizocilpine into the superficial part of subnucleus caudalis facilitated wind-up. The effect peaked at 25 min with a mean increase above control of 173+/-31%. Injection (0.5 microg) of either the less active enantiomer dizocilpine or saline into superficial subnucleus caudalis had no significant effect on subnucleus oralis convergent neurones. This suggests that NMDA-dependent interneurones, probably located in substantia gelatinosa of subnucleus caudalis, exert, in normal conditions, an inhibitory control on wind-up of convergent subnucleus oralis neurones. (2) The injection of larger doses (0.5 microg) into the superficial part of subnucleus caudalis induced a predominant inhibitory effect on wind-up. The mean peak effect at 15 min was 46+/-7% compared to control (100%). Small and large doses of dizocilpine injected into the deep part of subnucleus caudalis had a predominant inhibitory effect. The inhibition of wind-up of subnucleus oralis neurones after injection of NMDA receptor antagonists in superficial or deep subnucleus caudalis indicates that wind-up may be due, at least in part, to NMDA activation at synapses that do not involve the recorded convergent neurones.

Animals↗
PubMed2002

Investigation of ion transfer across the micro-water/nitrobenzene interface facilitated by a fullerene derivative.

A functionalized fullerene derivative containing a monoaza-18-crown-6 moiety was investigated by facilitated ion (such as Li+, Na+, K+, NH4+, Mg2+, and Ca2+) transfer across the micro-water/nitrobenzene interface supported at the tip of a micropipet. The current responses were detected by cyclic voltammetry and Osteryoung square wave voltammetry, which demonstrated that the facilitated ion transfer does occur by an interfacial complexation-dissociation process. The diffusion coefficient of this compound in nitrobenzene was approximately (5.90 +/- 0.04) x 10(-7) cm2 s(-1), which is 1 order of magnitude less than other common ionophores due to the large size of the molecule. The selectivity of this molecule toward the metal ions followed the sequence Na+ > Li+ > K+ > NH4+ > Ca2+ approximately equals Mg2+. In addition, this compound was also easy to form film at the water/nitrobenzene interface to inhibit the simple ion transfer of tetramethylammonium ion. However, the adsorption of this ionophore has less influence on the facilitated metal ion transfer.

Journal Article↗
PubMedDate not supplied

On the origin of intracellular compartmentation and organized metabolic systems.

The history of the development of the ideas and research of organized metabolic systems during last three decades is shortly reviewed. The cell cytoplasm is crowded with solutes, soluble macromolecules such as enzymes, nucleic acids, structural proteins and membranes. The high protein density within the large compartments of the cells predominantly determines the major characteristics of cellular environment such as viscosity, diffusion and inhomogeneity. The fact that the solvent viscosity of cytoplasm is not substantially different from the water is explained by intracellular structural heterogeneity: the intrinsic macromolecular density is relatively low within the interstitial voids in the cell because many soluble enzymes are apparently integral parts of the insoluble cytomatrix and are not distributed homogeneously. The molecular crowding and sieving restrict the mobility of very large solutes, binding severely restrict the mobility of smaller solutes. One of consequence of molecular crowding and hindered diffusion is the need to compartmentalize metabolic pathway to overcome diffusive barriers. Although the movement of small molecules is slowed down in the cytoplasm, the metabolism can successfully proceed and even be facilitated by metabolite channeling which directly transfers the intermediate from one enzyme to an adjacent enzyme without the need of free aqueous-phase diffusion. The enhanced probability for intermediates to be transferred from one active site to the other by sequential enzymes requires stable or transient interactions of the relevant enzymes, which associate physically in non-dissociable, static multienzyme complexes--metabolones, particles containing enzymes of a part or whole metabolic systems. Therefore, within the living cell the metabolism depends on the structural organization of enzymes forming microcompartments. Since cells contain many compartments and microenvironments, the measurement of the concentration of metabolites in whole cells or tissues gives an average cellular concentration and not that which is actually sensed by the active site of a specific enzyme. Thus, the microcompartmentation could provide a mechanism which can control metabolic pathways. Independently and in parallel to the developments described above, the ideas of compartmentation came into existence from the necessity to explain important physiological phenomena, in particular in heart research and in cardiac electrophysiology. These phenomena demonstrated the physiological importance of the biophysical and biochemical mechanisms described in this review.

Cell Compartmentation↗
PubMed1989

Embryonic versus adult myocardium: adenine nucleotide degradation during ischemia.

Neonatal myocardium demonstrates better recovery from ischemia than does adult tissue. We tested the hypothesis that developmental differences in adenine nucleotide degradation might facilitate recovery by quantitating depletion of high-energy phosphates in nine-day-old embryonic (n = 9) and 15-month-old adult (n = 14) chicken hearts at 15-, 30-, 45-, and 60-minute intervals of normothermic ischemia in vitro. Nucleotides adenosine triphosphate, adenosine diphosphate, and adenosine monophosphate and nucleosides adenosine, inosine, hypoxanthine, and xanthine were determined by high-performance liquid chromatography. Several observations in metabolite degradative response to ischemia were noted. The embryonic myocardium maintained higher adenosine triphosphate and adenosine monophosphate levels over the course of the investigation than did mature myocardium. Moreover, the adult group showed an increase in diffusible nucleoside pool metabolites. Relative immaturity of enzymes responsible for nucleotide degradation may facilitate postischemic recovery by preserving nondiffusible high-energy phosphate precursors to participate in salvage resynthesis of adenosine triphosphate.

Adenine Nucleotides↗
PubMed1995

Palmitate uptake by cardiac myocytes and endothelial cells.

The mechanisms regulating the cellular uptake of long-chain fatty acids are poorly understood. Although there is evidence that hepatocytes facilitate the uptake of ligands from the protein-bound fraction, it is not known whether cardiac myocytes also facilitate the uptake process. The present studies were designed to address the role of albumin in the uptake of long-chain fatty acids by cardiac myocytes isolated from adult male rats. At low albumin concentrations (1 microM), the myocyte palmitate clearance rate did not exceed that predicted by the diffusion-reaction model. At high albumin concentrations (300 and 600 microM), the clearance ratio test was used to determine whether myocytes facilitate the uptake of palmitate. As with the low albumin concentrations, the diffusion-reaction model accounted for the overall clearance rates. Because endothelial cells might be involved in enhancing fatty acid transport into myocytes, we also determined the effects of endothelial cells on palmitate uptake by cardiac myocytes. Based on cell number, the palmitate clearance rate by endothelial cells in the presence of albumin was only 7% of the cardiac myocyte clearance rate. Combining the endothelial cells with the myocytes did not result in any synergistic effect on the palmitate clearance rate.

Animals↗
PubMed2001

Detection, purification and characterisation of quorum-sensing signal molecules in plant-associated bacteria.

Quorum sensing (also called autoinduction) is a term that describes an environmental sensing system that allows bacteria to monitor their own population density. Autoinduction relies upon the interaction of a small diffusible signal molecule (the autoinducer) with a transcriptional activator protein to couple gene expression with cell population density. These signal molecules diffuse from bacterial cells and accumulate in the environment as a function of cell growth. Once a threshold concentration is reached, these signals serve as co-inducers to regulate the transcription of (a) set(s) of target genes. In Gram-negative bacteria, most autoinducers belong to the family of N-acylhomoserine lactones (AHLs). The detection of AHLs (or AHL-like activities) has been greatly facilitated by the development of sensitive bioassays that allow fast screening of microorganisms for diffusible signal molecules. AHL or diketopiperazine-mediated cell-cell signalling play roles in regulating different bacterial functions, such as antibiotic biosynthesis, production of virulence factors, exopolysaccharide biosynthesis, bacterial swarming, plasmid conjugal transfer and transition into the stationary phase. Several bacterial species that interact with plants produce AHL-like compounds. In this review, we will summarise the current knowledge about the detection, characterisation and purification of quorum-sensing molecules from plant-associated bacteria. We will also discuss some of the future prospects and biotechnological applications of autoinducers.

4-Butyrolactone↗
PubMed2000

Ca2+ dynamics at the frog motor nerve terminal.

Rises in free [Ca2+]i in response to various tetanic stimuli (Ca2+ transient) in frog motor nerve terminals were measured by recording fluorescence changes of Ca2+ indicators and analyzed in relation to short-term synaptic plasticity. Ca2+ transients reached a plateau after 10-20 impulses at 100 Hz and decayed in a three-exponential manner, in which the fast component was predominant. The plateau and fast component of the Ca2+ transient were elevated infralinearly with an increase in tetanus frequency. Computer simulation showed that the Ca2+ transients estimated from fluorescence changes faithfully reflect the true changes in [Ca2+]i except for the initial 20 ms. A slow Ca2+ chelator, EGTA, loaded into the nerve terminal, decreased the magnitude of both the fast and slow components of facilitation of transmitter release and the time constant of the former. A fast Ca2+ chelator, BAPTA, decreased the magnitude of fast facilitation but slightly increased its time constant. These results suggest that Ca2+ transients in the frog motor nerve terminals are primarily caused by Ca2+ entry and are dissipated by three components, in which the rate of the fast component is equivalent to that of free Ca2+ diffusion. The residual Ca2+ in the nerve terminals after stimulation accounts for the fast component of facilitation.

Animals↗
PubMed2006

Identification of diffuse and focal brain lesions by clinical magnetic resonance spectroscopy.

The purpose of this paper is to facilitate the comparison of magnetic resonance (MR) spectra acquired from unknown brain lesions with published spectra in order to help identify unknown lesions in clinical settings. The paper includes lists of references for published MR spectra of various brain diseases, including pyogenic abscesses, encephalitis (herpes simplex, Rasmussen's and subacute sclerosing panencephalitis), neurocysticercosis, tuberculoma, cysts (arachnoid, epidermoid and hydatid), acute disseminated encephalomyelitis (ADEM), adrenoleukodystrophy (ALD), Alexander disease, Canavan's disease, Krabbe disease (globoid cell leukodystrophy), Leigh's disease, megalencephalic leukoencephalopathy with cysts, metachromatic leukodystrophy (MLD), Pelizaeus-Merzbacher disease, Zellweger syndrome, HIV-associated lesions [cryptococcus, lymphoma, toxoplasmosis and progressive multifocal leukoencephalopathy (PML)], hydrocephalus and tuberous sclerosis. Each list includes information on the echo time(s) (TE) of the published spectra, whether a control spectrum is shown, whether the corresponding image and voxel position are shown and the patient ages if known. The references are listed in the approximate order of usefulness, based on spectral quality, number of spectra, range of echo times and whether the voxel positions are shown. Spectra of Zellweger syndrome, cryptococcal infection, toxoplasmosis and lymphoma are included, along with a spectrum showing propanediol (propylene glycol).

Brain Neoplasms↗

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