Autoradiographic localization of transported neutral amino acids in epithelia of cat submandibular and sublingual salivary glands

Summary Light-microscopic autoradiography was used to localize the cellular sites for neutral amino acid uptake in submandibular and sublingual salivary gland epithelia. The vasculature of isolated glands was perfused for 3–5 min with either L-(3-³H)serine or L-(4-³H)phenylalanine and then fixed by perfusion with buffered glutaraldehyde. In the submandibular gland the small neutral amino acid L-serine and the aromatic amino acid L-phenylalanine were localized to central acinar cells, demilunar cells and ductal cells. In the sublingual gland silver grains associated with each of these tritiated amino acids were localized to central acinar and ductal cells. Perfusion of both submandibular and sublingual glands with unlabelled L-serine (25 mM) or L-phenylalanine (30 mM) resulted in a significant decrease in the silver grain density associated with each labelled amino acid. The absence of silver grains in the lumina of acinar and ductal cells and the presence of tight junctions near the apical surface of the epithelium strongly suggest that the initial uptake of these amino acids was mediated by basolateral plasma membrane carriers.     

Autoradiographic Localization of Benzodiazepine Receptor Binding in Dissociated Cultures of Fetal Mouse Cerebral Cortex

Autoradiography utilizing photoaffinity labelling with [3H]flunitrazepam was used in living cultures of fetal mouse cerebral cortex in situ to localize benzodiazepine receptor binding sites. There was a predominant localization of silver grains over neurons; however, substantial labelling also occurred over nonneuronal background cells. Clonazepam (0.1 microM) and Ro 5-4864 (0.1 microM) displaced substantial numbers of silver grains over neurons and background cells, respectively. In addition, clonazepam displaced 58-68% of specific grains over background cells and Ro 5-4864 displaced 30% of grains over neurons, suggesting that multiple cell types in the CNS may participate in the neuropharmacologic actions of the benzodiazepines.     

A pyridine bridged dicarbene ligand and its silver(I) and palladium(II) complexes: synthesis, structures, and catalytic applications

The potentially tridentate ligand 2,6-bis[(3-methylimidazolium-1-yl)methyl]pyridine dibromide reacts readily with silver(I) oxide in dichloromethane or dimethylsulfoxide to give a dinuclear silver(I)-carbene complex that was isolated as the tetrafluoroborate salt. Single crystal X-ray crystallography shows that each silver(I) ion is bridged by two ligands bonding through the carbene donors. Treatment of the silver(I) complex with suitable palladium(II) precursors gave the complexes PdCl[(CNC)]BF₄ and [PdMe(CNC)]BF₄ (CNC=2,6-bis[(3-methylimidazolin-2-yliden-1-yl)methyl]pyridine), in which the pyridyl and both carbene moieties are coordinated to a single palladium(II). The palladium(II) complexes have been fully characterised, including X-ray crystallography, and exhibit good activities in the Heck coupling reaction of 4-bromoacetophenone and n-butyl acrylate.     

Fabrication of a Bionic Needle with both Super-Hydrophobic and Antibacterial Properties

Many biological surfaces possess unusual micro-nano hierarchical structures that could influence their wettability, which provide new methods for the construction of novel materials. In this work, silver nanoparticles were successfully coated on the surface of stainless steel needle by a simple electroless replacement reaction process between the AgNO₃ solution and the activated stainless steel needle. After the replacement reaction, porous micro/nanostructures were formed on the surface of the stainless steel needle. By modifying long chains of thiol molecules, the stainless steel needle exhibited good super-hydrophobic property with a contact angle greater than 150°. Moreover, the silver coated stainless steel needle (bionic needle) showed strong antibacterial activity against the gram-negative bacterium Escherichia coli (E. coli). By calculating the area of the inhibition zone against E. coli formed on agar medium, the antibacterial activity of the bionic needle with the contact angle of 152° is much better than that with the contact angle of 138°. The as-prepared bionic needle with both super-hydrophobic and antibacterial properties has the potential to be applied in modern medical devices.     

Dynamic Mechanisms of the Bactericidal Action of an Al2O3-TiO2-Ag Granular Material on an Escherichia coli Strain

The bactericidal activity of an Al₂O₃-TiO₂-Ag granular material against an Escherichia coli strain was confirmed by a culture-based method. In particular, 100% of microorganisms were permanently inactivated in 30 to 45 min. The present work aimed to investigate the mechanisms of the bactericidal action of this material and their dynamics on Escherichia coli using different techniques. Observations by transmission electron microscopy (TEM) at different times of disinfection revealed morphological changes in the bacteria as soon as they were put in contact with the material. Notably highlighted were cell membrane damage; cytoplasm detachment; formation of vacuoles, possibly due to DNA condensation, in association with regions exhibiting different levels of electron density; and membrane lysis. PCR and flow cytometry analyses were used to confirm and quantify the observations of cell integrity. The direct exposure of cells to silver, combined with the oxidative stress induced by the reactive oxygen species (ROS) generated, was identified to be responsible for these morphological alterations. From the first 5 min of treatment with the Al₂O₃-TiO₂-Ag material, 98% of E. coli isolates were lysed. From 30 min, cell viability decreased to reach total inactivation, although approximately 1% of permeable E. coli cells and 1% of intact cells (10⁵ genomic units · ml⁻¹) were evidenced. This study demonstrates that the bactericidal effect of the material results from a synergic action of desorbed and supported silver. Supported silver was shown to generate the ROS evidenced.     

Antibacterial and antibiofilm effects of silver nanoparticles against the uropathogen Escherichia coli U12

The drug-resistant bacterial strains' emergence increases day by day. This may be a result of biofilm presence, which protects bacteria from antimicrobial agents. Thus, new approaches must be used to control biofilm-related infections in healthcare settings. In such a study, biological silver nanoparticles were introduced in such a study as an anti-biofilm agent against multidrug-resistant E. coli U12 on urinary catheters. Seven different silver nanoparticles concentrations were tested for their antimicrobial activities. Also, anti-biofilm activities against E. coli U12 were tested. Using the dilution method, the silver nanoparticles concentration of 85 μg/ml was the MIC (Minimum Inhibitory Concentration) that had excellent biocompatibility and showed significant antibacterial activity against E. coli U12. Scanning electron microscopy (SEM) confirmed that the highest efficient dose of silver nanoparticles was 340 μg/ml at 144 h that reduced adhesion of E. coli U12 to the urinary catheter. E. coli U12 cells ruptured cell walls and cell membranes after being examined using transmission electron microscopy (TEM). Thus, biologically prepared silver nanoparticles could be used to coat medical devices since it is effective and promising to inhibit biofilm formation by impregnating urinary catheters with silver nanoparticles.     

Increased production of L-serine in Escherichia coli through Adaptive Laboratory Evolution

L-serine is a promising building block biochemical with a high theoretical production yield from glucose. Toxicity of L-serine is however prohibitive for high-titer production in E. coli. Here, E. coli lacking L-serine degradation pathways was evolved for improved tolerance by gradually increasing L-serine concentration from 3 to 100 g/L using adaptive laboratory evolution (ALE). Genome sequencing of isolated clones revealed multiplication of genetic regions, as well as mutations in thrA, thereby showing a potential mechanism of serine inhibition. Additional mutations were evaluated by MAGE combined with amplicon sequencing, revealing role of rho, lrp, pykF, eno, and rpoB on tolerance and fitness in minimal medium. Production using the tolerant strains resulted in 37 g/L of L-serine with a 24% mass yield. The resulting titer is similar to the highest production reported for any organism thereby highlighting the potential of ALE for industrial biotechnology.     

Spontaneous glycosylation of glycosaminoglycan substrates by adherent fibroblasts.

SV40-transformed mouse fibroblasts migrate upon, and spontaneously glycosylate, plastic substrates derivatized with chondroitin-6 sulfate and hyaluronic acid. Autoradiography of cultures prelabeled with 3H-glucose and 3H-galactose demonstrates the presence of silver grains near adherent cells. Silver grains are particularly dense near cells cultured on chondroitin sulfate. No significant grains are observed on control dishes or dishes derivatized with polygalacturonic acid. Radioactive material left by cells is not removed by boiling the dishes in 8 M urea and 10% sodium dodecylsulfate, suggesting that it is covalently linked to the derivatized plastic. Acid hydrolysis shows that the radioactive material consists of glucuronic acid and N-acetylglucosamine when the prelabeled cells are cultured on hyaluronic acid. When cells are cultured on chondroitin sulfate, the radioactive product consists only of glucuronic and and N-acetylgalactosamine sulfate. Sonicates of prelabeled cells or the supernatants from cultures of intact prelabeled cells add no SDS-urea-resistant radioactivity to dishes derivatized with these glycosaminoglycans.     

Biochemical Studies of Olfaction: Binding Specificity of Odorants to a Cilia Preparation from Rainbow Trout Olfactory Rosettes

Cilia isolated from the olfactory epithelium (olfactory rosettes) of rainbow trout (Salmo gairdneri) bind amino acids, which are odor stimuli to this species. We demonstrate that L-threonine, L-serine, and L-alanine bind to a common site, TSA, in the cilia preparation. All possible mixtures of two of the amino acids as competitors, with the third as the 3H-labeled ligand, were studied. The effect of two combined (unlabeled) competitors was always substantially less than additive compared with their actions singly. Along with additional inhibition studies using mixtures of inhibitors, the data show that the three odorants must interact with at least one common binding site, TSA. Binding of L-[3H]lysine to site L was unaffected by addition of L-threonine, L-serine, or L-alanine, establishing its independence from site TSA. L-Arginine inhibited binding of L-[3H]lysine, showing that both of these basic amino acids interact with site L. The data establish the presence, in trout olfactory cilia, of at least two separate and noninteracting populations of odorant binding sites, TSA and L.     

The effect of L-2-amino-4-methoxy-trans-3-butenoic acid on serine hydroxymethyl transferase

The tumour growth inhibitor L-2-amino-4-methoxy-trans-3-butenoic acid (Ro07-7957) inhibits serine hydroxymethyltransferase in cytosolic extracts of Walker carcinoma non-competitively with respect to L-serine with an apparent inhibition constant similar to the K_m-value for L-serine. The kinetics of inactivation suggest that it reacts as an irreversible substrate analogue. Incubation of Walker cells with Ro07-7957 causes an increase in serine hydroxymethyltransferase activity which is most pronounced at concentration ≤LD₅₀. This increase in enzyme activity does not occur in the presence of cycloheximide. These results suggest that inhibition of serine hydroxymethyltransferase in intact cells is accompanied by an increase in enzyme biosynthesis and that the growth inhibitory property of Ro07-7957 does not involve interference with the conversion of serine to glycine.     

Localization of newly-synthesized DNA in a mammalian cell as visualized by high resolution autoradiography

The localization of newly-replicated DNA in mouse cells of line P815 in culture is studied by high resolution autoradiography. After 20 or 30 sec of incorporation of ${}^3\text{H-thymidine}$, the silver grains are found throughout the nucleus with a relatively higher (about two-fold) density over the peripheral region of the nucleus. After a further 1 h of chase with non-radioactive thymidine, or after 19 h of continuous growth with the radioactive precursor, the pattern of the nuclear distribution of label is not appreciably different from that found after a short pulse. Autoradiography, combined with EDTA staining resulting in a preferential bleaching of the chromatin, reveals after a short pulse of ${}^3\text{H-thymidine}$ that most of the silver grains lie close to, or over, the border zone between condensed chromatin and the interchromatin region. The results are discussed in the context of other recent findings concerning the sites of DNA replication in eukaryotic cells obtained with different cell systems.     

Quantitative studies on the localization of the cholinergic receptor protein in the normal and denervated electroplaque from Electrophorus electricus

Electroplaques dissected from the electric organ of Electrophorus electricus are labeled by tritiated alpha1-isotoxin from Naja nigricollis, a highly selective reagent of the cholinergic (nicotinic) receptor site. Preincubation of the cell with an excess of unlabeled alpha-toxin and with a covalent affinity reagent or labeling in the presence of 10(-4) M decamethonium reduces the binding of [3H]alpha- toxin by at least 75%. Absolute surface densities of alpha-toxin sites are estimated by high-resolution autoradiography on the basis of silver grain distribution and taking into account the complex geopmetry of the cell surface. Binding of [3H]alpha-toxin on the noninnervated face does not differ from background. Labeled sites are observed on the innervated membrane both between the synapses and under the nerve terminals but the density of sites is approx. 100 times higher at the level of the synapses than in between. Analysis of the distance of silver grains from the innervated membrane shows a symmetrical distribution centered on the postsynaptic plasma membrane under the nerve terminal. In extrasynaptic areas, the barycenter of the distribution lies approximately 0.5 micrometer inside the cell, indicating that alpha-toxin sites are present on the membrane of microinvaginations, or caveolae, abundant in the extrajunctional areas. An absolute density of 49,600 +/- 16,000 sites/micrometer2 of postsynaptic membrane is calculated; it is in the range of that found at the crest of the folds at the neuromuscular junction and expected from a close packing of receptor molecules. Electric organs were denervated for periods up to 142 days. Nerve transmission fails after 2 days, and within a week all the nerve terminals disappear and are subsequently replaced by Schwann cell processes, whereas the morphology of the electroplaque remains unaffected. The denervated electroplaque develops some of the electrophysiological changes found with denervated muscles (increases of membrane resting resistance, decrease of electrical excitability) but does not become hypersensitive to cholinergic agonists. Autoradiography of electroplaques dissected from denervated electric organs reveals, after labeling with [3H]alpha- toxin, patches of silver grains with a surface density close to that found in the normal electroplaque. The density of alpha-toxin binding sites in extrasynaptic areas remains close to that observed on innervated cells, confirming that denervation does not cause an increase in the number of cholinergic receptor sites. The patches have the same distribution, shape,and dimensions as in subneural areas of the normal electroplaque, and remnants of nerve terminal or Schwann cells are often found at the level of the patches. They most likely correspond to subsynaptic areas which persist with the same density of [3H]alpha-toxin sites up to 52 days after denervation. In the adult synapse, therefore, the receptor protein exhibits little if any tendency for lateral diffusion.     

Studies on the release of barley aleurone cell proteins: Autoradiography

Summary Both uptake and incorporation of radioactivity from [³H]l-leucine into gibberellic-acid (GA₃)-treated aleurone layers of barley (Hordeum vulgare L.) was enhanced by pretreatment with 5 mM potassium bromate. The effect of 5 mM KBrO₃ on amino-acid incorporation was quantitative rather than qualitative and could be partly reversed by the addition of neutralized casein hydrolysate at 10 mg/ml. Autoradiographs of GA₃-treated aleurone cells pulsed with [³H]leucine showed distribution of silver grains predominantly over the endoplasmic reticulum (ER) and aleurone grains. After chasing with carrier l-leucine for 60 min, fewer silver grains were associated with the ER and aleurone grains while nearly half of the silver was associated with the ground cytoplasm of the cell. Autoradiographs were prepared from aleurone cells previously stratified by ultracentrifugation. After a 10-min pulse of label, the silver grains were found over the central ER zone of centrifuged cells; however, with an increase in duration of the chase, label was found distributed throughout the aleurone grain and spherosome region of the cell. The silver grains which were located over the central zone of centrifuged cells at the end of the pulse were almost exclusively associated with the ER. There is no evidence for association of label with dictyosomes or with vesicles derived from dictyosomes. The experimental evidence indicates that labelled amino acids are incorporated into aleurone cells on the ER and are released from these cells without the participation of a membrane-bound vesicle.     

Does the Antibacterial Activity of Silver Nanoparticles Depend on the Shape of the Nanoparticle? A Study of the Gram-Negative Bacterium Escherichia coli

In this work we investigated the antibacterial properties of differently shaped silver nanoparticles against the gram-negative bacterium Escherichia coli, both in liquid systems and on agar plates. Energy-filtering transmission electron microscopy images revealed considerable changes in the cell membranes upon treatment, resulting in cell death. Truncated triangular silver nanoplates with a {111} lattice plane as the basal plane displayed the strongest biocidal action, compared with spherical and rod-shaped nanoparticles and with Ag⁺ (in the form of AgNO₃). It is proposed that nanoscale size and the presence of a {111} plane combine to promote this biocidal property. To our knowledge, this is the first comparative study on the bactericidal properties of silver nanoparticles of different shapes, and our results demonstrate that silver nanoparticles undergo a shape-dependent interaction with the gram-negative organism E. coli.     

<Emphasis Type="Italic">glyA</Emphasis> gene knock-out in <Emphasis Type="Italic">Escherichia coli</Emphasis> enhances L-serine production without glycine addition

In E. coli, glyA encodes for serine hydroxymethyltransferase (SHMT), which converts L-serine to glycine. When engineering L-serine-producing strains, it is therefore favorable to inactivate glyA to prevent L-serine degradation. However, most glyA knockout strains exhibit slow cell growth because of the resulting lack of glycine and C₁ units. To overcome this problem, we overexpressed the gcvTHP genes of the glycine cleavage system (GCV), to increase the C₁ supply before glyA was knocked out. Subsequently, the kbl and tdh genes were overexpressed to provide additional glycine via the L-threonine degradation pathway, thus restoring normal cell growth independent of glycine addition. Finally, the plasmid pPK10 was introduced to overexpress pgk, serA ^Δ197 , serC and serB, and the resulting strain E4G2 (pPK10) accumulated 266.3 mg/L of L-serine in a semi-defined medium without adding glycine, which was 3.18-fold higher than the production achieved by the control strain E3 (pPK10). This strategy can accordingly be applied to disrupt the L-serine degradation pathway in industrial production strains without causing negative side-effects, ultimately making L-serine production more efficient.     

Macromolecular synthesis in the livers of aging mice as revealed by electron microscopic radioautography

For the purpose of studying the aging changes of macromolecular synthesis in animal cells, we studied many groups of aging mice during development and aging from fetal day 19 to postnatal newborn, juvenile, young adults, aged and senescent adults up to 12 and month 24 (2 years). They were injected with ³H-thymidine, ³H-uridine or ³H-leucine, precursors for DNA, RNA and proteins, as well as ³H-glucose, ³H-glucosamine, ³⁵S-sulfuric acid, or ³H-glycerol for glucide and lipid precursors, respectively, then sacrificed and the liver tissues were taken out, fixed and processed for light and electron microscopic radioautography. On many radioautograms the localization of silver grains demonstrating DNA, RNA and proteins in hepatocytes in respective aging groups were analyzed qualitatively. The number of silver grains and the number of cell organelles in each cell of each animal in respective aging groups were analyzed quantitatively in relation to the aging of individual animals. The results revealed that the localization of respective precursors as well as the number of silver grains in cell nuclei, cell organelles, changed with the aging of animals. The numbers of labeled nuclei and cell organelles, as well as the numbers of silver grains in nuclei and cell organelles changed due to aging of individual animals. The number of mitochondria, the number of labeled mitochondria and the mitochondrial labeling index labeled with silver grains were counted in each hepatocyte. It was demonstrated that the numbers of mitochondria, the numbers of labeled mitochondria and the labeling indices showing DNA, RNA and protein synthesis at various ages from embryonic day 19 to postnatal newborn day 1, 3, 9, 14, adult month 1, 2 and 6, reaching the maxima, then decreased to senile year 1 to 2, indicating the aging changes. The results indicated that mitochondria in hepatocytes synthesized nucleic acids and proteins independently from the nuclei, but their synthetic activities were affected from the aging of the individual animals.     

Rapid and steady-state amino acid transport in perfused human fibroblasts and colon adenocarcinoma cells: effects of methotrexate

Initial and steady-state uptakes of serine and phenylalanine by human fibroblasts and human colon tumour cells were studied applying a double isotope dilution technique to perfused populations of cultivated cells retained on microcarrier beads. This new method permits the differentiation of the unidirectional transport parameters and can also distinguish between membrane-associated processes and independently intracellular events in isolated cells. High initial L-serine uptake values in colon adenocarcinoma cells became negative under steady-state conditions. To determine if the observed negative L-serine uptake was produced by the rapid efflux of intracellular L-[3H]serine, the cells were treated with methotrexate (MTX) (an inhibitor of cytosolic dihydrofolate reductase). The modified curve of L-[3H]serine uptake after MTX treatment suggests that, under these experimental conditions, net serine transport is non concentrative in colon tumour cells and could be modulated by the rate of intracellular serine metabolism; it also suggests that MTX does not directly affect serine transport in perfused human colon adenocarcinoma cells. Initial and steady-state uptakes of phenylalanine were high in both fibroblasts and tumour cells and were unaffected by MTX treatment.     

Polysaccharide-based silver nanoparticles synthesized by <Emphasis Type="Italic">Klebsiella oxytoca</Emphasis> DSM 29614 cause DNA fragmentation in <Emphasis Type="Italic">E. coli</Emphasis> cells

Silver nanoparticles (AgNPs), embedded into a specific exopolysaccharide (EPS), were produced by Klebsiella oxytoca DSM 29614 by adding AgNO₃ to the cultures during exponential growth phase. In particular, under aerobic or anaerobic conditions, two types of silver nanoparticles, named AgNPs-EPS^aer and the AgNPs-EPS^anaer, were produced respectively. The effects on bacterial cells was demonstrated by using Escherichia coli K12 and Kocuria rhizophila ATCC 9341 (ex Micrococcus luteus) as Gram-negative and Gram-positive tester strains, respectively. The best antimicrobial activity was observed for AgNPs-EPS^aer, in terms of minimum inhibitory concentrations and minimum bactericidal concentrations. Observations by transmission electron microscopy showed that the cell morphology of both tester strains changed during the exposition to AgNPs-EPS^aer. In particular, an electron-dense wrapped filament was observed in E. coli cytoplasm after 3 h of AgNPs-EPS^aer exposition, apparently due to silver accumulation in DNA, and both E. coli and K. rhizophila cells were lysed after 18 h of exposure to AgNPs-EPS^aer. The DNA breakage in E. coli cells was confirmed by the comparison of 3-D fluorescence spectra fingerprints of DNA. Finally the accumulation of silver on DNA of E. coli was confirmed directly by a significant Ag⁺ release from DNA, using the scanning electrochemical microscopy and the voltammetric determinations.     

Sequential changes in glycolipid expression during human B cell differentiation: enzymatic bases

We have previously reported that human B cell differentiation is accompanied by sequential changes in glycosphingolipid expression. Pre-B cells contain lacto-series type II chain-based glycolipids and GM3 ganglioside; mature/activated B cells do not synthesize lacto-series compounds but express GM3 and globo-series glycolipids (Gb3 and Gb4); terminally differentiated B cells, in addition to these compounds, also contain GM2 ganglioside. At the cell surface, Gb3, Gb4 and GM2 constitute stage-specific antigens. To elucidate the biosynthetic mechanism leading to these modifications we have compared activities of the glycosyltransferases involved in the core structure assembly and the first elongation steps of neo-lacto, ganglio- and globo-series glycolipids. These glycosyltransferase activities have been measured in B cell lines and normal B lymphocytes at various stages of differentiation. We first determined the optimal requirements of the four glycosyltransferases which wynthesize Lc3, GM3, Gb4 and GM2 glycolipids in B lymphocytes and then tested these enzymes and the Gb3 synthetase in the selected B cells. The following results were obtained: ß1 → 3N-Acetylglucosaminyltransferase (Lc3 synthetase) has a high activity in pro- and pre-B cells whereas it is undetectable in more differentiated cells; α2 → 3 sialyltransferase (GM3 synthetase) is activated from the pre-B cell stage to the terminally differentiated myeloma cells; α → 4 galactosyltransferase (Gb3 synthetase) is only detected in cells representing the late stages of B cell differentiation; ß1 → 3N-Acetylgalactosaminyltransferase (Gb4 synthetase) is only found in some lymphoblastoid cell lines, representative of activated B cells whereas the ß1 → 4 N-Acetylgalactosaminyltransferase (GM2 synthetase) has a high activity in these lymphoblastoid cell lines and in terminally differentiated myeloma cells. These results suggest that the sequential shifts in the three major glycosphingolipid series observed during B cell differentiation are mostly due to sequential activations of the corresponding glycosyltransferases.