Template-directed synthesis and selective adsorption of oligoadenylates on hydroxyapatite

Summary Polyuridylic acid is adsorbed completely from aqueous solution by hydroxyapatite under conditions that permit template-directed synthesis of oligoadenylates in free solution. The yield of oligoadenylates is enhanced to almost the same extent by poly(U) in the presence or the absence of hydroxyapatite. Under very similar conditions small quantities of hydroxyapatite adsorb higher molecular-weight oligoadenylates selectively from a mixture of oligomers.On the basis of these results we propose a mechanism for prebiotic oligonucleotide formation in which selective adsorption on hydroxyapatite or some other immobilized anion-exchanging material plays a major role. Monomers are released from the surface for reactivation, while oligomers are retained in a protected environment by adsorption to the apatite surface.Abbreviations poly(U) polyuridylate - HA hydroxyapatite - ImpA adenosine 5-phosphorimidazolide - pA adenosine 5-phosphate - ADP adenosine 5-diphosphate - MeIm 1-methylimidazole - EDTA ethylenediaminetetraacetic acid - (pA)_n (n = 2, 3, 4) oligoadenylates with 5-terminal phosphates The nature of the internucleotide linkages is not specifiedThe nature of the internucleotide linkages is not specified     

Isolation of mutants ofLactobacillus bacterias,grown in skim milk,that resist fatty acid inhibition

The effect of saturated fatty acids from 60 to 160 and oleic acid was determined onLactobacillus leichmannii growing in skim milk. The growth of this strain was markedly inhibited by fatty acids from 80 to 120 but not by straight chain fatty acids greater than 130 or less than 70 and oleate. Laurate was the fatty acid with the highest bactericidal effect. Similar results, with little changes depending on strains, were obtained withL. casei, L. plantarum, L. bulgaricus, L. lactis, L. helveticus. Mutants from theseLactobacillus organisms, resistant to fatty acid inhibition, were isolated by a recycling selection procedure. These mutants exhibited high levels of oxidation for laurate. The presence of 2 mM of this compound in the skim milk culture increased the fatty acid oxidation activity four to ten times higher than was exhibited by the parent strains. The practical implications of these observations are discussed.     

Parameters affecting attachment ofBacillus thuringiensis var.israelensis toxin to latex beads

Summary Bacillus thuringiensis var.israelensis protein toxin radiolabeled with¹⁴C-iodoacetamide was bound to latex beads. The efficiency of attachment was not affected by the temperature or length of incubation or by the presence of several different buffers, salts, or organic solvents. However, attachment decreased dramatically at pH values >8.5 or in the presence of detergents (anionic, neutral, or cationic). Protein concentrations 2–3 mg/m² of bead surface led to a progressively decreasing efficiency of protein adsorption. With minor variations, these findings should also be applicable to the attachment of numerous other proteins of diagnostic significance.Abbreviations BSA bovine serum albumin - Bti Bacillus thuringiensis var.israelensis - EDTA ethylenediaminetetraacetic acid - HEPES N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic acid - LAT latex agglutination test - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecylsulfate - TCA trichloroacetic acid - Tris Tris(hydroxymethyl)aminomethane - TTAB tetradecyltrimethylammonium bromide     

Adsorption of bacteriophage on bacterial surface and on the surface of Hg dropping electrode

Summary The influence of the ionic strength of the medium on the adsorption of bacteriophage T 2 to the surfaces of a mercury dropping electrode on one hand and ofbacteria E. coli B on the other hand was studied. The adsorption on the mercury surface was determined by measurement of the differential capacity of the electrode double layer, the adsorption to bacteria was estimated from the decrease of free phage particles in a bacterial suspension with time. The adsorption to the mercury electrode increases with increasing ionic strength of the medium, but adsorption to the surface of bacteria increases at first, has a maximum at concentrations between 0,1 to 0,5 M and decreases with further increase of ionic strength. The decrease of adsorption of phage to the bacterial surface is assumed to be caused by the blocking of specific sites on the bacterial surface by adsorbed ions which sterically prevent the adsorption of the phage. Such specific sites are not present on the electrode surface, therefore adsorption increases further with increasing ionic strength probably due to the neutralization of surface charges of the phage and of the electrode. The saturated surface-concentration of the phage _s was calculated from the dependence of the differential capacity on the concentration. It is concluded from _s value obtained that the phage particles are scattered with wide intervals on the electrode surface with a degree of coverage of approximately 140.Abbreviations used DNA deoxyribonucleic acid - N Avogadro number The authors wishes to express their gratitude to the late Prof.Ferdinand Hercík, director of the Institute of Biophysics, for the initiation of this work and stimulating interest. The authors are also indebted to Dr. J.Koudelka for his kind gift of phage T 2 sample and to Dr. M.Vízdalová for her valuable comments during preparation of this article.     

Specific inhibition of tetrapyrrole biosynthesis by 3-amino 2,3-dihydrobenzoic acid (gabaculin) in the cyanobacteriumSynechococcus 6301

Gabaculin (3-amino 2,3-dihydrobenzoic acid) inhibited the growth of cyanobacteria but not of other prokaryotes. Exposure of growing cultures ofSynechococcus 6301 to 50 M gabaculin resulted in an immediate and complete inhibition of the synthesis of chlorophylla and phycocyanin. With 8 M gabaculin, tetrapyrrole synthesis was suppressed for approximately 10 h and then resumed at a lower rate than in untreated organisms. The effect of 50 M gabaculin was reversed by transferring organisms to inhibitor-free medium; chlorophylla synthesis began within 5 h and exponential growth was re-established after about 25 h. Compared with 4,6-dioxoheptanoic acid (DA) and laevulinic acid (LA), gabaculin was a much more potent inhibitor of tetrapyrrole synthesis inSynechococcus 6301. The catalytic activity of -aminolaevulinic acid (ALA) dehydratase in vitro was inhibited by DA and LA but not by 1 mM gabaculin. However, the specific activity of the dehydratase was much lower in organisms exposed to the inhibitor for 36 h. Growing cultures and cell suspensions ofSynechococcus 6301 exposed to DA excreted appreciable quantities of ALA. In contrast, relatively small amounts of ALA accumulated in the presence of gabaculin alone and this inhibitor blocked the excretion of ALA caused by DA. This suggests that the primary effect of gabaculin is the specific inhibition of the C₅ pathway for the biosynthesis of ALA.Abbreviations ALA -aminolaevulinic acid - DA 4,6-dioxoheptanoic acid - LA laevulinic acid - GABA -aminobutyric acid     

Expression of surface hydrophobicity encoded by R-plasmids in Escherichia coli laboratory strains

The inclusion of drug-resistance plasmids (R-plasmids) in Escherichia coli strains has been shown to determine the formation of specific surface structures which could modify bacterial surface characteristics relevant for pathogenic processes.Thirtyone R-plasmids (from different incompatibility groups) have been transferred to three E. coli laboratory strains, and surface hydrophobicity modifications have been measured by three methods: salting-out, adsorption to hexadecane and adsorption to xylene.The results obtained show that the presence of R-plasmids produced variations which are dependent on the receptor strains and measuring method employed. Also, it has been found that the plasmids behave differently depending on the strain in which they are included.The results obtained by the salting-out method are not correlative with those obtained by adsorption to hydrocarbons, probably due to the implication of different hydrophobic molecules in the interaction with salt or hydrocarbons.Concluding, the choice of receptor strain and measuring method are of great importance for the investigation of surface hydrophobicity (and probably other characteristics) encoded by R-plasmids.Abbreviations TSB Trypticase soya broth - TSA trypticase soya agar     

Production of gluconic acid and 2-ketogluconic acid by Klebsiella aerogenes NCTC 418

2-Ketogluconic acid and, to a lesser extent, gluconic acid were found to be major products of glucose catabolism by phosphate-limited cultures of Klebsiella aerogenes NCTC 418, and together accounted for up to 46% of the glucose carbon that was metabolized.Although the concentrations of both acids increased sub-stantially at low growth rates, their specific rates of synthesis decreased markedly, as did the proportion of glucose converted into these products.Determination of the affinity constant, for glucose, of phosphate-limited organisms showed it to be not significantly different from that of glucose-limited organisms (K _s 50 M), indicative of the phosphotransferase uptake system. And since these organisms possessed an active glucose 6-phosphate dehydrogenase, and had no detectable glucose dehydrogenase activity, it was concluded that gluconic acid and 2-ketogluconic acid arose from their corresponding phosphorylated metabolites, and not directly from glucose.     

Scanning electron microscopy of the gut microflora of two earthworms: Lumbricus terrestris and Octolasion cyaneum

Scanning electron microscopy was used to investigate the presence of microorganisms, probably bacteria, on the gut surface of earthworms. The washed surfaces of the intestines of two earthworms, Lumbricus terrestris and Octolasion cyaneum, were examined. Numerous organisms resembling bacteria were observed throughout the gut, some in situations suggesting attachment. Compared with similar investigations in other invertebrates, there were fewer bacteria, showing less morphological diversity, on the earthworm gut surface. The majority of organisms viewed were coccoid, some were filamentous, and a few rod-shaped cells were observed. Cocci, often in chains, were seen in the foregut of both species. Although cocci were also numerous in the midgut region, particularly in the typhlosole, in O. cyaneum tufts of segmented, filamentous organisms were also seen with some segments resembling spores. Fewer organisms were found in the hindgut, but in L. terrestris there were segmented, filamentous organisms, attached to the epithelium by way of a socket-like structure, similar to that by which segmented, filamentous bacteria (SFBs) are attached to the ileum of rats and mice. Transmission electron microscopy of the hindgut of L. terrestris was undertaken to explore the structure and attachment of SFBs to the gut epithelium. However, although a few rod-shaped bacteria were observed, no SFBs were located. The observations reported here provide evidence that earthworms have an attached gut microflora of filamentous microorganisms which are probably indigenous, and as far as we are aware this is the first published report of such findings in these invertebrates. Offprint requests to: J.M. Jolly.     

Cloning and expression of a member of the Aspergillus niger gene family encoding alpha-galactosidase.

Summary An enzyme with -galactosidase activity and an apparent molecular weight of 82 kDa was purified from culture medium of Aspergillus niger. The N-terminal amino acid sequence of the purified protein shows similarity to the N-terminal amino acid sequence of -galactosidases from several other organisms. Oligonucleotides, based on the N-terminal amino acid sequence, were used as probes to clone the corresponding gene from a EMBL3 gene library of A. niger. The cloned gene (aglA) was shown to be functional by demonstrating that the 82 kDa -galactosidase is absent from a strain with a disruption of the agIA gene, and is over-produced in strains containing multiple copies of the aglA gene. Enzyme activity assays revealed that the 82 kDa -galactosidase A represents a minor extracellular -galactosidase activity in A. niger.     

Patterns of intramolecular carbon isotopic heterogeneity within amino acids of autotrophs and heterotrophs

A survey of the intramolecular C isotopic composition of a variety of organisms was conducted to investigate the potential of intramolecular isotopic measurements as a tracer of biological or geochemical processes. Based on a consideration of inorganic C sources and enzymatic fractionations, contrasting predictions were made for the relative ¹³C enrichments of the -carboxyl carbons fixed by the anapleurotic ()-carboxylation pathway during amino acid synthesis by photoautotrophs and heterotrophs. To test the model predictions, the stable C isotopic compositions of the acid hydrolyzable C fraction, the total amino acid -carboxyl C fraction and the -carboxyl C of glutamate from a variety of autotrophic and heterotrophic organisms were compared. The relative ¹³C enrichments of carboxyl carbons in the bulk amino acid fraction and in glutamate conformed qualitatively to model predictions. Macroalgal taxa possessed a significantly less enriched carboxyl C fraction than did either C3 or C4 vascular plants, indicating the presence of a different -carboxylation pathway operating in these organisms. In most multicellular heterotrophs, the isotopic composition of the amino acid carboxyl carbons closely resembled that of their food sources. Amino acids are apparently assimilated into tissue proteins directly from their diets without significant metabolic modification. However, shifts in the isotopic composition of the carboxyl C fractions in some organisms were detected that were consistent with the occurrence of significant resynthesis of amino acids from non-amino acid precursors. Comparison of plant leaves and roots provided evidence of environmentally controlled assimilate partitioning. Intramolecular isotopic measurements of biological molecules provide unique insights into the origins and transformations of bio-molecules.     

Molecular Modelling of a Multiphosphorylated Sequence Motif Bound to Hydroxyapatite Surfaces

Proteins and peptides containing the multiphosphorylated motif -Ser(P)-Ser(P)- Ser(P)--Glu-Glu- stabilise amorphous calcium phosphate (ACP) in body fluids and bind with high affinity to crystalline calcium phosphate phases such as hydroxyapatite (HA) regulating crystal growth. Binding of this motif to hydroxyapatite surfaces was investigated in this study using molecular modelling techniques. Using a three-step computational procedure, we have determined the relative binding energies of the motif Ser(P)-Ser(P)-Ser(P)-Glu-Glu to different crystalline surfaces of HA. This analysis revealed preferences of the motif for (100) and (010) surfaces of the crystal and preferences for particular orientations on a given surface. These preferences are principally governed by electrostatic interactions between the crystal lattice and the peptide with the most stable conformers adopting structures where alternate residues exhibit backbone angles characteristic of a -strand and values of an -helix or a distorted -helix, allowing maximal interaction between the acidic side groups and surface calciums. The results of this study are consistent with experimentally-derived data on the interaction of multiphosphorylated proteins/peptides with HA and have implications for the role of these proteins/peptides in calcium phosphate stabilisation and biomineralisation processes.Electronic Supplementary Material available.     

Serolis luethjei n.sp., a new isopod crustacean from the Weddell Sea

Summary The spatial distribution and size-dependence of oxygen consumption (respiration) and production by microplankton in near surface waters of the Canadian Arctic were measured during summer, 1983. High oxygen flux rates (consumption and production) were observed near surface (upper 20–30 m) and were generally associated with high phytoplankton biomass (chlorophyll a) levels. A substantial portion of the respiration (>50%), however, was below the euphotic zone. Integrated oxygen fluxes (0–100 m) were approximately in balance (i.e., net oxygen production 0) at most locations sampled. In general, oxygen fluxes were higher than have been observed in the Southern Ocean but in the same range as found in temperate coastal waters. Size-fractionation studies showed that most (>60%) of the oxygen production and phytoplankton biomass (chlorophyll a) were associated with organisms greater than 35 m. On the other hand, more than 70% of the respiration was associated with organisms less than 35 m; on average, more than 50% of the respiration was associated with organisms less than 1 m. These results are consistent with theoretical studies and with experimental observations from temperate waters.     

Free energies of amino acid adsorption on silica in neutral aqueous medium as estimated from high-performance liquid-chromatographic retention data

Summary Equilibrium constants (K) and free energies (–G) of amino acid adsorption on silica in a neutral aqueous medium were calculated from the retention values measured by means of high-performance liquid chromatography on a silica gel column. For most amino acids (with the exception of proline) –G values were negative andK < 1, thus showing very low adsorption. Influence of the structure of the-substituent on adsorbability is analyzed. A linear dependence of –G on the number of aliphatic carbon atoms was shown for the series: glycine-alanine-valine-leucine-isoleucine.Abbreviations Gly glycine - Ala alanine - Pro proline - Val valine - Ile isoleucine - Leu leucine - Ser serine - Thr threonine - Cys cysteine - Asn asparagine - Gln glutamine - Asp aspartic acid - Glu glutamic acid - Met methionine - His histidine - Phe phenylalanine - Tyr tyrosine - DOPA 3,4-dioxyphenylalanine - Trp tryptophan     

Effect of keratin on the lipid composition of Scopulariopsis brevicaulis (Bainier)

Lipid content and composition of Scopulariopsis brevicaulis cultured with or without its natural substrate keratin was investigated. Lipid content was found to be lower in the presence of keratin (maximum 12% against 19% in the absence of keratin), with differences in levels of linoleic acid (C₁₈₌₂), palmitic (C₆₀) and to a lesser extent palmitoleic (C₁₆₌₁) and -linolenic (C₁₈₌₃) acids. The degree of unsaturation was correspondingly lower in the organisms cultured with keratin as substrate.     

Decrease in cell surface sialic acid in etoposide-treated Jurkat cells and the role of cell surface sialidase

The present study investigated the mechanism underlying alterations of cell surface sugar chains of Jurkat cells by inducing apoptosis with etoposide, an inhibitor of topoisomerase II. Within 3[emsp4 ]h of etoposide treatment, flowcytometric analysis revealed a decrease in Maackia amurensis agglutinin recognized 2,3-linked sialic acid moieties and an increase in Ricinus communis agglutinin recognized galactose. The results suggested that asialo-sugar chains on glycoconjugates were rapidly induced on the etoposide-treated cell surface. To clarify the desialylation mechanism, we studied 2,3-sialyltransferase mRNA expression and the activity of sialidase on the cell surface during etoposide-induced apoptosis. The expression of hST3Gal III and hST3Gal IV mRNAs were down-regulated and sialidase activity on the cell surface increased threefold within 2[emsp4 ]h of etoposide treatment. Moreover, the decrease in 2,3-linked sialic acid levels was significantly suppressed in the presence of 2,3-dehydro-2-deoxy-N-acetylneuraminic acid, an inhibitor of sialidase. These results suggested that activation or exposure of sialidase on the cell surface was induced by etoposide treatment and was the main cause of the decrease in sialic acids.     

Rapid and simple purification of a novel extracellular β-amylase from Bacillus sp.

Bacillus sp. KYJ 963, a local isolate, produced an extracellular amylase with M _r=59 kDa. The amylase was easily purified by adsorption on soluble starch. The analyses of TLC and N-terminal amino acid sequence from the purified protein revealed that the enzyme was a novel -amylase which could not hydrolyze maltose or -cyclodextrin and its N-terminal amino acid sequence was A-V-N-G-Q-S-F-N-S-N-Y-K-T-Y-K-.     

Ultrastructure of “Gordona aurantiaca” Tsukamura 1971

Ultrastructure of Gordona aurantiaca^* M 296 (8128) was studied after the lead citrate coloration, whereas the cell envelope architecture was investigated by ruthenium red staining for outer wall acidic polysaccharides and the periodic-acid-thiocarbohydrazide-silver-proteinate cytochemical procedure (Thiéry method) for the detection of 1-2 glycol bond containing polysaccharides. The ultrastructural morphology of bacteria was distinct from both the mycobacteria and nocardia. The bacilli had a typical gram-positive cell wall that contained a thin, uniformly distributed, polysaccharide outer layer (POL) at its surface. The Thiéry cytochemical method stained only the cytoplasmic membrane, but not the cell wall, a feature that is common to the mycolic acid containing theCorynebacterium-Mycobacterium-Nocardia (CMN) group of organisms. The negative staining of the unfixed preparations of bacilli showed ribbonlike surface structures, common to the CMN group of organisms. The electron-microscopic preparations showed numerous lysing bacilli with bacteriophages indicating that the strain used was lysogenic.     

A laser-T-jump study of the adsorption of dipolar molecules to planar lipid membranes

The adsorption of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) as well as of other dipolar molecules to the interface of artificial lipid membranes gives rise to a change of the dipole potential between the membrane interior and water. As a consequence of the adsorption of the neutral species, the conductance of planar membranes, observed in the presence of the macrocyclic ion carriers nonactin or valinomycin, may change by many orders of magnitude. Using this effect in combination with a laser-T-jump technique, the kinetics of the adsorption process were measured and were interpreted on the basis of a Langmuir-isotherm. A partition coefficient (at small concentrations) of _HA =4.7·10^–4 cm, a rate constant of desorption k _HA100 s^-1 and a maximum surface density N _D=7.7·10¹³/cm² were found. The concentration at half saturation is K _HA=2.7·10^-4 M.Using these values the membrane conductance induced by the ion carrier nonactin and the shape of the current-voltage relationship as a function of the ligand concentration in water was analyzed. A maxiumum dipole potential of V _D ^max =-239 m V and a contribution of b=3.1·10^-15V cm² per single adsorbed 2,4-D molecule was found. 74% of the dipole potential acts on the inner membrane barrier separating the two interfacial adsorption planes of nonactin. The remainder (26%) favours interfacial complex formation between nonactin and K⁺ from the aqueous phase. The data hold for membranes formed from dioleoyllecithin in n-decane.     

Differential expression of sialylglycoconjugates and sialidase activity in distinct morphological stages of<Emphasis Type="Italic"> Fonsecaea pedrosoi</Emphasis>

The expression of sialoglycoconjugates in Fonsecaea pedrosoi conidia, mycelia, and sclerotic cells was analyzed using influenza A and C virus strains, sialidase treatment, and lectin binding. Conidium and mycelium whole cells were recognized by Limax flavus (LFA), Maackia amurensis (MAA), and Sambucus nigra (SNA) lectins, denoting the presence of surface sialoglycoconjugates containing 2,3- and 2,6-sialylgalactosyl sequences. Sialidase-treated conidia reacted more intensively with peanut agglutinin (PNA), confirming the occurrence of sialyl-galactosyl linkages. Conidial cells agglutinated in the presence of influenza A and C virus strains, which confirmed the results obtained from lectin-binding experiments and revealed the presence of sialoglycoconjugates bearing 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac₂) surface structures. Western blotting analysis with peroxidase-labeled LFA demonstrated the occurrence of sialylglycoproteins in protein extracts from conidia and mycelia, with molecular masses corresponding to 56 and 40 kDa. An additional band of 77 kDa was detected in conidial extracts, suggesting an association between sialic acid expression and morphogenesis. Synthesis of sialic acids was correlated with sialidase expression, since both conidial and mycelial morphological stages presented secreted and cell-associated enzyme activity. Sialoglycoconjugates were not detected in F. pedrosoi sclerotic cells from in vitro and in vivo sources, which also do not express sialidase activity. The surface sialyl residues in F. pedrosoi are apparently involved in the fungal interaction with immune effector cells, since sialidase-treated conidia were less resistant to phagocytosis by human neutrophils from healthy individuals. These findings suggest that sialic acid expression in F. pedrosoi varies according to the morphological transition and may protect infecting propagules against immune destruction by host cells.     

Preliminary investigations on somatic embryogenesis from leaf discs of red oak (Quercus rubra L.)

Somatic embryos were obtained from leaf discs of juvenile red oak plants. Basal inductive nutrient medium was a modified Murashige and Skoog solution enriched with 500 mg L^–1 casein hydrolysate, 100 mg L^–1 polyvinylpyrrolidone, 5.4 M naphthaleneacetic acid and 0.09 M benzyladenine. Embryogenesis was obtained only from leaf discs in the presence of light and increased when the adaxial surface of the explants (with midrib or main veins present) was in contact with the medium. Large variation was observed in all experiments. Recurrent embryogenesis was observed at the base of embryo clusters with callus present; conversely, embryogenic potential was rapidly lost by subculturing full calli. Maturation, germination and development of isolated somatic embryos were obtained. However, the vast majority of embryos did not have viable apical bud meristems and on only a few occasions were shoots produced.Abbreviations BA N⁶-benzyladenine - CH casein hydrolysate - 2iP isopentenyladenine - NAA naphthaleneacetic acid - 2.4-D 2.4-dichlorophenoxyacetic acid - GA gibberellic acid - PVP polyvinylpyrrolidone