VULNERABILITY OF THE IMMATURE BRAIN TO PHENYLACETATE INTOXICATION: TISSUE PERMEABILITY TO PHENYLACETATE

In the rat phenylacetate readily penetrates into such tissues as brain, eyes, heart, kidneys, and liver. One hour after the subcutaneous injection of sodium phenylacetate into rats ranging in age from 1 to 65 days, the ratios of the concentrations (μmolig) in blood/tissue were approx 1:1 in all of these tissues with the exception of the brain. The blood-brain ratio changed from 1:1 in the 1–7 day old rat to the adult ratio of 2:1 at 21 days of age. The free access of phenylacetate into the immature brain is especially relevant to phenylketonuria, a metabolic disease in which excessive amounts of phenylacetate are produced in peripheral tissues.     

METABOLISM OF TISSUE CULTURES : III. A METHOD FOR MEASURING THE PERMEABILITY OF TISSUE CELLS TO SOLUTES

By using radioactive isotopes in tissue cultures, the rate of permeation of substances into cells can be measured independently of concurrent metabolic reactions of these substances. Techniques of obtaining and analyzing data are described. Examples are given using radioactive potassium and phosphorus. Using cultures of chick embryo muscle, turnover time for cell potassium is 6 hours, and for cell inorganic phosphate is 7 hours in the examples cited. Permeability rates, based on estimates of the cell surface involved and expressed as millimoles per cm.² per hour, are of the order of magnitude of 10^–6 for potassium and of 10^–7 for phosphate.     

EPITHELIAL COLLAGENS AND GLYCOSAMINOGLYCANS IN THE EMBRYONIC CORNEA : Macromolecular Order and Morphogenesis in the Basement Membrane

The embryonic corneal epithelium synthesizes both collagen and chondroitin sulfate and excretes them across the basement membrane into the subepithelial space where they assemble into a spiraling orthogonal matrix of fibrils. The assembly of collagen into fibrils is first apparent at the outer face of the basement membrane in a region of ordered chondroitin sulfate molecules. Hyaluronate, another morphogenetically important corneal macromolecule, is produced at these early stages only by the inner endothelium. These correlated biosynthetic and ultrastructural data demonstrate discrete macromolecular products of the two corneal epithelia with differing morphogenetic properties and functions.     

STUDIES ON PERMEABILITY OF MEMBRANES : VI. MENSURATION OF THE DRIED COLLODION MEMBRANE (CALCULATION OF DIMENSIONS AND OF RELATIONS TO CERTAIN BIOLOGICAL MEMBRANES).

The flat type of dried collodion membrane used by Michaelis and his associates in numerous investigations has been subjected to mensuration in order that the dimensions of these membranes may be placed on record. The membranes had a functioning area of about 30 cm., were approximately 0.1 mm. in thickness and were composed on the average of 87 per cent by volume of collodion and 13 per cent by volume of pores. In reviewing some of the previously reported results of diffusion experiments with non-electrolytes in the light of the calculated values for the total pore area for the same membranes additional evidence was presented to show that a smaller molecule (acetone) probably utilizes a much larger percentage of the total pore area for its diffusion than is available for a larger molecule (glycerol). By using the figures of Fricke and McClendon for the thickness of the membrane of the red blood cell some comparisons were drawn between the dried collodion membrane as a model for certain biological membranes and the red blood cell membrane. In these comparisons emphasis was placed on the exaggerated importance of small electromotive forces and very slight permeabilities when these were associated with membranes of such extreme thinness as the red blood cell membrane.     

Characterization of the Blood-Brain Barrier: Protein Composition of the Capillary Endothelial Cell Membrane

Microvessels were isolated from canine cerebral cortex, and the composition of the endothelial cell membrane was investigated. Endothelial cell membranes were separated from the surrounding basement membrane, solubilized, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 12% gels. Staining with Coomassie Blue revealed a characteristic banding pattern of at least 12 major proteins with apparent molecular weights between 14,000 and 250,000. When proteins from red blood cell ghosts were run simultaneously, no similarities were observed, except for proteins at apparent molecular weights of 43,000 (band 3) and 35,000 (band 4). These two proteins migrated exactly to the positions of the erythrocyte proteins actin and glyceraldehyde 3-phosphate dehydrogenase, respectively. Membrane glycoproteins in gels were also examined by the use of fluorescent lectins. Of the fluorescein isothiocyanate-conjugated (FITC) lectins tested, only FITC-concanavalin A had an affinity for any membrane components. Diazotized [125I]iodosulfanilic acid, a membrane-impermeable reagent, was used to label the internal (lumen) cell surface and the external (antilumen) cell surface. Autoradiography and determination of radioactivity levels in gel slices showed that several proteins were specifically labeled, and that major differences in radioactivity of proteins existed in internal and external labeling experiments. It is concluded that the protein composition of the luminal membrane is different from that of the antiluminal membrane.     

Identification of the Multidrug Resistance-Related Membrane Glycoprotein as an Acceptor for Cyclosporine

Abstract

The immunosuppressive agent cyclosporine A (CSA) has been shown to reverse multidrug resistance (MDR) in malignant cells. In the present study, a ³H-cyclosporine diazirine analogue (³H-PL-CS) was used to photolabel viable MDR cells. The 170 kDa membrane P-glycoprotein, which functions as a drug efflux pump, was strongly labeled. The binding of ³H-cyclosporine diazirine analogue to P-glycoprotein was competable by excess cyclosporine A and by the nonimmunosuppressive cyclosporine H. These results suggest that cyclosporine reverses the MDR phenotype by binding directly to P-glycoprotein and that this binding is not dependent on the immunosuppressive potential of the cyclosporine derivative. The identification of P-glycoprotein as a cyclosporine binding protein has obvious implications for cancer chemotherapy.     

Identification of the Plasma Membrane Proteolipid Protein as a Constituent of Brain Coated Vesicles and Synaptic Plasma Membrane

We have analyzed brain coated vesicles and synaptic plasma membrane for the presence of the plasma membrane proteolipid protein. Coated vesicles were isolated from calf brain gray matter with a final purification on Sephacryl S-1000 and reisolated twice by chromatography to ensure homogeneity. Fractions were analyzed by gel electrophoresis, immunoblotting for clathrin heavy chain, and by electron microscopy. Using an immunoblotting assay we were able to demonstrate the presence of the plasma membrane proteolipid protein in these coated vesicles at a significant level (i.e., approximately 1% of the bilayer protein of these vesicles). Reisolation of coated vesicles did not diminish the concentration of the protein in this fraction. Removal of the clathrin coat proteins or exposure of the coated vesicles to 0.1 M Na2CO3 showed that the plasma membrane proteolipid protein is not removed during uncoating and lysis but is intrinsic to the membrane bilayer of these vesicles. These studies demonstrate that plasma membrane proteolipid protein represents a significant amount of the bilayer protein of coated vesicles, suggesting that these vesicles may be a transport vehicle for the intracellular movement of the plasma membrane proteolipid protein. Isolation of synaptic plasma membranes proteolipid adult rat brain and estimation of the plasma membrane proteolipid protein content using the immunoblotting method confirmed earlier studies that show this protein is present in this membrane fraction at high levels as well (approximately 1-2%). The level of this protein in the synaptic plasma membrane suggests that the synaptic plasma membrane is one major site to which these vesicles may be targeted or from which the protein is being retrieved.     

Chilling Susceptibility of the Blue-green Alga Anacystis nidulans: II. STIMULATION OF THE PASSIVE PERMEABILITY OF CYTOPLASMIC MEMBRANE AT CHILLING TEMPERATURES

Potassium ions and amino acids were found to leak from the cytoplasm to the outer medium when the blue-green alga, Anacystis nidulans, was exposed to the chilling temperatures. The leakage was marked below the critical temperature regions, the midpoint values for which were around 5 and 14 C in cells grown at 28 and 38 C, respectively. These temperature regions coincided with those critical for the susceptibility of the photosynthetic activities and the carotenoid absorption spectrum previously studied (Ono TA, N Murata 1981 Plant Physiol 67: 176-181).     

Coronavirus Particle Assembly: Primary Structure Requirements of the Membrane Protein

Coronavirus-like particles morphologically similar to normal virions are assembled when genes encoding the viral membrane proteins M and E are coexpressed in eukaryotic cells. Using this envelope assembly assay, we have studied the primary sequence requirements for particle formation of the mouse hepatitis virus (MHV) M protein, the major protein of the coronavirion membrane. Our results show that each of the different domains of the protein is important. Mutations (deletions, insertions, point mutations) in the luminal domain, the transmembrane domains, the amphiphilic domain, or the carboxy-terminal domain had effects on the assembly of M into enveloped particles. Strikingly, the extreme carboxy-terminal residue is crucial. Deletion of this single residue abolished particle assembly almost completely; most substitutions were strongly inhibitory. Site-directed mutations in the carboxy terminus of M were also incorporated into the MHV genome by targeted recombination. The results supported a critical role for this domain of M in viral assembly, although the M carboxy terminus was more tolerant of alteration in the complete virion than in virus-like particles, likely because of the stabilization of virions by additional intermolecular interactions. Interestingly, glycosylation of M appeared not essential for assembly. Mutations in the luminal domain that abolished the normal O glycosylation of the protein or created an N-glycosylated form had no effect. Mutant M proteins unable to form virus-like particles were found to inhibit the budding of assembly-competent M in a concentration-dependent manner. However, assembly-competent M was able to rescue assembly-incompetent M when the latter was present in low amounts. These observations support the existence of interactions between M molecules that are thought to be the driving force in coronavirus envelope assembly.     

COMPOSITION OF CELLULAR MEMBRANES IN THE PANCREAS OF THE GUINEA PIG : I. Isolation of Membrane Fractions

The subcellular components involved in the synthesis, transport, and discharge of secretory proteins in the guinea pig pancreatic exocrine cell have been isolated from gland homogenates by differential and gradient centrifugation. They include rough and smooth microsomes derived respectively from the rough endoplasmic reticulum and Golgi periphery, a zymogen granule fraction consisting mainly of mature zymogen granules and a smaller population of condensing vacuoles, and a plasmalemmal fraction. Membrane subfractions were obtained from the particulate components by treatment with mild (pH 7.8) alkaline buffers which extract the majority (>95%) of the content of secretory proteins, allowing the membranes to be recovered from the extracting fluid by centrifugation. The purity of the fractions was assessed by electron microscopy and by assaying marker enzymes for cross-contaminants. The rough and smooth microsomes were essentially free of mitochondrial contamination; the smooth microsomes contained <15% rough contaminants. The zymogen granule fraction and its derived membranes were free of rough microsomes and contained <3% contaminant mitochondria. The plasmalemmal fraction was heterogeneous as to origin (deriving from basal, lateral, and apical poles of the cell) and contained varying amounts of adherent fibrillar material arising from the basement membrane and terminal web. The lipid and enzymatic composition of the membrane fractions are described in the following reports.     

THE NUCLEUS OF NOCTILUCA SCINTILLANS : Aspects of Nucleocytoplasmic Exchanges and the Formation of Nuclear Membrane

The unicellular organism, Noctiluca, has been examined with the electron microscope. The nucleus is small compared to the very large size of the cell, but the nuclear border has an organization which indicates an active nucleocytoplasmic exchange. Whereas annuli are missing over most parts of the nuclear membrane proper, there are "annulated vesicles" in a layer inside the nuclear membrane. The hypothesis is put forth that nuclear substances move through the annuli into these vesicles, and that the annulated vesicles themselves are transported through the nuclear membrane. The various forms of the annulated vesicles are consistent with this hypothesis. An implication of this postulate is the synthesis of annulated membranes inside a closed nucleus which are physically separate from the endoplasmic reticulum. The chromosomes are in a state resembling prophase chromosomes and are surrounded by granular masses. Only a small portion of the entire nuclear volume is occupied by the chromosomes. There are many nucleolus-like bodies.     

THE BIOGENESIS OF MITOCHONDRIA : III. The Lipid Composition of Aerobically and Anaerobically Grown Saccharomyces cerevisiae as Related to the Membrane Systems of the Cells

The growth conditions known to influence the occurrence of mitochondrial profiles and other cell membrane systems in anaerobic cells of S. cerevisiae have been examined, and the effect of the several growth media on the lipid composition of the organism has been determined. The anaerobic cell type containing neither detectable mitochondrial profiles nor the large cell vacuole may be obtained by the culture of the organism on growth-limiting levels of the lipids, ergosterol, and unsaturated fatty acids. Under these conditions, the organism has a high content of short-chain saturated fatty acids (10:0, 12:0), phosphatidyl choline, and squalene, compared with aerobically grown cells, and it is especially low in phosphatidyl ethanolamine and the glycerol phosphatides (phosphatidyl glycerol + cardiolipin). The high levels of unsaturated fatty acids normally found in the phospholipids of the aerobic cells are largely replaced by the short-chain saturated acids, even though the phospholipid fraction contains virtually all of the small amounts of unsaturated fatty acid present in the anaerobic cells. Such anaerobic cells may contain as little as 0.12 mg of ergosterol per g dry weight of cells while the aerobic cells contain about 6 mg of ergosterol per g dry weight. Anaerobic cell types containing mitochondrial profiles can be obtained by the culture of the organism in the presence of excess quantities of ergosterol and unsaturated fatty acids. Such cells have increased levels of total phospholipid, ergosterol, and unsaturated fatty acids, although these compounds do not reach the levels found in aerobic cells. The level of ergosterol in anaerobic cells is markedly influenced by the nature of the carbohydrate in the medium; those cells grown on galactose media supplemented with ergosterol and unsaturated fatty acids have well defined mitochondrial profiles and an ergosterol content (2 mg per g dry weight of cells) three times that of equivalent glucose-grown cells which have poorly defined organelle profiles. Anaerobic cells which are low in ergosterol synthesize increased amounts of squalene.     

New Antibiotic Molecules: Bypassing the Membrane Barrier of Gram Negative Bacteria Increases the Activity of Peptide Deformylase Inhibitors

Background

Multi-drug resistant (MDR) bacteria have become a major concern in hospitals worldwide and urgently require the development of new antibacterial molecules. Peptide deformylase is an intracellular target now well-recognized for the design of new antibiotics. The bacterial susceptibility to such a cytoplasmic target primarily depends on the capacity of the compound to reach and accumulate in the cytosol.

Methodology/Principal Findings

To determine the respective involvement of penetration (influx) and pumping out (efflux) mechanisms to peptide deformylase inhibitors (PDF-I) activity, the potency of various series was determined using various genetic contexts (efflux overproducers or efflux-deleted strains) and membrane permeabilizers. Depending on the structure of the tested molecules, two behaviors could be observed: (i) for actinonin the first PDF-I characterized, the AcrAB efflux system was the main parameter involved in the bacterial susceptibility, and (ii), for the lastest PDF-Is such as the derivatives of 2-(5-bromo-1H-indol-3-yl)-N-hydroxyacetamide, the penetration through the membrane was a important limiting step.

Conclusions/Significance

Our results clearly show that the bacterial membrane plays a key role in modulating the antibacterial activity of PDF-Is. The bacterial susceptibility for these new antibacterial molecules can be improved by two unrelated ways in MDR strains: by collapsing the Acr efflux activity or by increasing the uptake rate through the bacterial membrane. The efficiency of the second method is associated with the nature of the compound.     

Coral Luminescence Identifies the Pacific Decadal Oscillation as a Primary Driver of River Runoff Variability Impacting the Southern Great Barrier Reef

The Pacific Decadal Oscillation (PDO) is a large-scale climatic phenomenon modulating ocean-atmosphere variability on decadal time scales. While precipitation and river flow variability in the Great Barrier Reef (GBR) catchments are sensitive to PDO phases, the extent to which the PDO influences coral reefs is poorly understood. Here, six Porites coral cores were used to produce a composite record of coral luminescence variability (runoff proxy) and identify drivers of terrestrial influence on the Keppel reefs, southern GBR. We found that coral skeletal luminescence effectively captured seasonal, inter-annual and decadal variability of river discharge and rainfall from the Fitzroy River catchment. Most importantly, although the influence of El Niño-Southern Oscillation (ENSO) events was evident in the luminescence records, the variability in the coral luminescence composite record was significantly explained by the PDO. Negative luminescence anomalies (reduced runoff) were associated with El Niño years during positive PDO phases while positive luminescence anomalies (increased runoff) coincided with strong/moderate La Niña years during negative PDO phases. This study provides clear evidence that not only ENSO but also the PDO have significantly affected runoff regimes at the Keppel reefs for at least a century, and suggests that upcoming hydrological disturbances and ecological responses in the southern GBR region will be mediated by the future evolution of these sources of climate variability.     

THE SUBMITOCHONDRIAL LOCALIZATION OF MONOAMINE OXIDASE : An Enzymatic Marker for the Outer Membrane of Rat Liver Mitochondria

Controlled osmotic lysis (water-washing) of rat liver mitochondria results in a mixed population of small vesicles derived mainly from the outer mitochondrial membrane and of larger bodies containing a few cristae derived from the inner membrane. These elements have been separated on Ficoll and sucrose gradients. The small vesicles were rich in monoamine oxidase, and the large bodies were rich in cytochrome oxidase. Separation of the inner and outer membranes has also been accomplished by treating mitochondria with digitonin in an isotonic medium and fractionating the treated mitochondria by differential centrifugation. Treatment with low digitonin concentrations released monoamine oxidase activity from low speed mitochondrial pellets, and this release of enzymatic activity was correlated with the loss of the outer membrane as seen in the electron microscope. The low speed mitochondrial pellet contained most of the cytochrome oxidase and malate dehydrogenase activities of the intact mitochondria, while the monoamine oxidase activity could be recovered in the form of small vesicles by high speed centrifugation of the low speed supernatant. The results indicate that monoamine oxidase is found only in the outer mitochondrial membrane and that cytochrome oxidase is found only in the inner membrane. Digitonin treatment released more monoamine oxidase than cytochrome oxidase from sonic particles, thus indicating that digitonin preferentially degrades the outer mitochondrial membrane.     

The Organic Acid Metabolism of Cox's Orange Pippin Apples: I. SOME EFFECTS OF THE ADDITION OF ORGANIC ACIDS TO THE PEEL OF THE FRUIT

1. The basic respirations (CO₂-output and O₂-uptake) of Cox'sOrangePippin apples and of the peel tissue prepared from themwerecompared in fruit in various stages of development, bothinitiallyand after storage at 12°C. Both show the samegeneral trend,although as the apples become mature the peakvalue of the respirationclimacteric tends to rise in the wholefruit and fall in thepeel.
2. The effect of adding malate orcitrate on the respiration ofthe same samples of peel was studied.
3. Three broad stages of development were observed. During thefirst stage (petal fall to 60 days after) the metabolic patternappears to be different from the two later stages. Here O₂-uptakeas well as C₂-output are influenced by the addition of bothmalate and, to a considerably less extent, citrate. In stage2 (60–125 days from petal fall), the malate effect (CO₂-output)is small until after detachment from the tree, when it risessharply. In stage 3 (125 days to full maturity) the malate effectfollows the course expected for earlier work, namely, it developsat the same time as the climacteric rise in respiration. Thepossible reasons for the different behaviour of the peel atthe three stages is discussed.
4. Results were similar in generaltrend for Cox's Orange Pippinapples grown on different rootstocksand under different culturalconditions.
5. It is suggested thatthe malate effect is most active in theepidermal and hypodermaltissues of the fruit.

     

Preliminary Studies on Membrane Filtration for the Production of Potable Water: A Case of Tshaanda Rural Village in South Africa

Ultrafiltration (UF) systems have been used globally for treating water from resources including rivers, reservoirs, and lakes for the production of potable water in the past decade. UF membranes with a pore size of between 0.1 and 0.01 micrometres provide an effective barrier for bacteria, viruses, suspended particles, and colloids. The use of UF membrane technology in treating groundwater for the supply of potable water in the impoverished and rural village, Tshaanda (i.e., the study area) is demonstrated. The technical and administrative processes that are critical for the successful installation of the pilot plant were developed. Given the rural nature of Tshaanda, the cultural and traditional protocols were observed. Preliminary results of the water quality of untreated water and the permeate are presented. Escherichia coli in the untreated water during the dry season (i.e., June and July) was 2 cfu/100 ml and was <1 cfu/100 ml (undetected) following UF, which complied with the WHO and South African National Standards and Guidelines of <1 cfu/100 ml. During the wet/rainy season (February) total coliform was unacceptably high (>2419.2 cfu/100 ml) before UF. Following UF, it dramatically reduced to acceptable level (7 cfu/100 ml) which is within the WHO recommended level of <10 cfu/100 ml. Additionally, during the wet/rainy season E. coli and enterococci were unacceptably high (40.4 cfu/100 ml and 73.3 cfu/100 ml, respectively) before UF but were completely removed following UF, which are within the WHO and SANS recommended limit. The values for electrical conductivity (EC) and turbidity were constantly within the WHO recommended limits of 300 µS/cm corrected at 25°C and <5 NTU, respectively, before and after UF, during dry season and wet season. This suggests that there is no need for pre-treatment of the water for suspended particles and colloids. Considering these data, it can be concluded that the water is suitable for human consumption, following UF.