Pesticinogeny: a Characteristic Useful for Presumptive Identification and Isolation of Pasteurella pestis

Current methods of identifying Pasteurella pestis rely heavily on tests specific for detecting fraction I, the envelope antigen. Pesticin I, a bacteriocin inhibitory for P. pseudotuberculosis, has been demonstrated in nearly all tested strains isolated from human infections. The results of using this characteristic as an identifying trait for P. pestis were compared with results reported for detecting fraction I by fluorescent-antibody and antiserum-agar techniques. Data indicate that, although certain atypical strains of P. pestis fail to react in one system or the other, a combination of these tests provides positive identification in all cases. Detection of P. pestis in contaminated materials is greatly facilitated, and the simplicity of this test makes it a valuable tool in the study of plague infections and an important adjunct to methods currently in use. The use of the pesticin I assay is not intended to replace other accepted techniques, but rather to supplement them and increase the effectiveness of plague investigation.     

Pesticins. 3. Expression of coagulase and mechanism of fibrinolysis

Mutational loss of pesticin I, a bacteriocin-like substance produced by Pasteurella pestis, is known to result in concomitant loss of a coagulase and fibrinolytic factor. No relationship was detected between pesticinogeny and other tested properties either associated with virulence or peculiar to P. pestis. Pesticin I was distinguished from the coagulase and fibrinolytic activities on the basis of anatomical distribution, behavior during gel filtration, and sensitivity to heat. Coagulase and the fibrinolytic factor were not differentiated by these criteria. Spontaneous suppressor mutations causing reversion to pesticinogeny were not detected, nor were such mutants obtained by treatment with ultraviolet light or 2-aminopurine. Attempts to demonstrate a common activator of pesticin I, coagulase, or the fibrinolytic factor in extracts of pesticinogenic cells were not successful. These results are in accord with the hypothesis that at least two structural genes for the three activities reside on a replicon distinct from the chromosome proper. Fibrinolytic activity was significantly reduced in the presence of 0.003 m epsilon-aminocaproic acid and was nonexistent on fibrin films freed from endogenous plasminogen by treatment with heat. Fibrinolytic activity on heated films could be restored by addition of plasma or serum from six mammalian species. Accordingly, the plague fibrinolytic factor, like staphylokinase or urokinase, promotes the conversion of plasminogen to plasmin.     

Photosystem I from the unusual cyanobacterium Gloeobacter violaceus

Photosystem I (PS I) from the primitive cyanobacterium Gloeobacter violaceus has been purified and characterised. Despite the fact that the isolated complexes have the same subunit composition as complexes from other cyanobacteria, the amplitude of flash-induced absorption difference spectra indicates a much bigger antenna size with about 150 chlorophylls per P700 as opposed to the usual 90. Image analysis of the PS I preparation from Gloeobacter reveals that the PS I particles exist both in a trimeric and in a monomeric form and that their size and shape closely resembles other cyanobacterial PS I particles. However, the complexes exhibit a higher molecular weight as could be shown by gel filtration. The preparation contains novel polypeptides not related to known Photosystem I subunits. The N-terminal sequence of one of those polypeptides has been determined and reveals no homology to known or hypothetical proteins. Immunoblotting shows a cross-reaction of three of the polypeptide bands with an antibody raised against the major LHC from the diatom Cyclotella cryptica. Electron microscopy reveals a novel T-shaped complex which has never been observed in any other cyanobacterial PS I preparation. 77 K spectra of purified PS I show an extreme blue-shift of the fluorescence emission, indicating an unusual organisation of the PS I antenna system in Gloeobacter. This revised version was published online in June 2006 with corrections to the Cover Date.     

The action of interferon preparations on strains of Legionella of various serogroups and species

The action of different preparations of interferon on Legionella strains has been studied in vivo and in vitro. The preparations of leukinferon at a concentration of 500 international units (I.U.) and reaferon at a concentration of 10,000 I.U. have been found to produce an inhibiting effect on Legionella strains in vitro, in a medium with carbon-yeast agar. Leukinferon at a concentration of 125 I.U. suppresses the growth of L. pneumophila also in a liquid medium. The preparation of leukinferon at a minimal concentration of 100 I.U. has been found to suppress the development of lethal infection in chick embryos infected with L. pneumophila strain Philadelphia 1.     

Synthesis of 2-alkoxy-8-hydroxyadenylpeptides: towards synthetic epitope-based vaccines

The preparation of three different 2-alkoxy-8-hydroxyadenylpeptide conjugates has been accomplished by solid-phase synthesis combined with 'on-resin' Cu(I) catalyzed Huisgen cycloaddition. The immunogenicity of the compounds has been evaluated in IL-12 production and antigen presentation assays.     

Biochemical studies of taste sensation. IX. Enhancement of L-[3H]glutamate binding to bovine taste papillae by 5'-ribonucleotides

The interaction of the taste substance monosodium L-glutamate with taste receptors has been investigated. Binding of L-[3H]glutamate was measured to preparations of bovine circumvallate (taste) papillae (type I preparation) and to control tongue epithelial preparations (type II preparation) devoid of taste receptors. Binding is operationally defined using a membrane filtration assay. Substantially greater binding occurred to the type I preparation than to the type II preparation, and the binding to the type I preparation showed evidence of saturation. The apparent Kd of L-glutamate was estimated to be in the range of 20--30 mM. The unique taste effect of L-glutamate was considered to depend importantly on its demonstrated synergism in combination with certain 5'-ribonucleotides. A several-fold enhancement of binding of L-[3H]glutamate occurred in the presence of certain 5'-ribonucleotides. 5'-GMP, 5'-IMP and 5'-UMP each increased the binding of L-[3H]glutamate, while 5'-XMP, 5'-AMP and 5'-CMP did not. None of these nucleotides affected the lower level of binding to the type II preparation. Neither the free bases, adenine and guanine, their nucleosides nor their di- or triphosphonucleotides were effective in increasing L-[3H]glutamate binding to the type I preparation. The nucleotide specificity of the glutamate binding enhancement therefore shows a marked similarity with the nucleotide specificity in evoking the synergistic taste effect in humans.     

A cellular factor involved in the formation of a DNA-synthesizing complex from DNA polymerase I in Escherichia coli

A factor (‘E’) has been identified which stabilizes an endogenous DNA-synthesizing complex involving DNA polymerase I. The complex is separated from free DNA polymerase by polyacrylamide gel electrophoresis. The factor will reform the complex after it has been dissociated and will convert a preparation of DNA polymerase I to complex. The factor and the DNA-synthesizing complex both appear to be localized at the cell membrane.     

Isolation of hexokinase II isoenzyme from the hyaloplasm of rat skeletal muscle

A method for obtaining partially purified preparation of II isozyme of hexokinase from cytosol of the rat skeletal muscles has been suggested. According to the data of electrophoretic and kinetic analyses the preparation does not practically contain I isozyme of hexokinase and is characterized by high enzymatic activity. The obtained preparation of II isozyme of hexokinase may be successfully used for research of the adsorption mechanism controlling the enzyme activity.     

Phosphoenolpyruvate-dependent protein kinase enzyme I of Streptococcus faecalis: purification and properties of the enzyme and characterization of its active center

Enzyme I, the phosphoenolpyruvate:protein phosphotransferase (EC 2.7.3.9), which is part of the bacterial phosphoenolpyruvate- (PEP) dependent phosphotransferase system, has been purified from Streptococcus faecalis by using a large-scale preparation. Size exclusion chromatography revealed a molecular weight of 140 000. On sodium dodecyl sulfate gels, enzyme I gave one band with a molecular weight of 70 000, indicating that enzyme I consists of two identical subunits. The first 59 amino acids of the amino-terminal part of the protein have been sequenced. It showed some similarities with enzyme I of Salmonella typhimurium. The active center of enzyme I has also been determined. After phosphorylation with [32P]PEP, the enzyme was cleaved by using different proteases. Labeled peptides were isolated by high-performance liquid chromatography on a reversed-phase column. The amino acid composition or amino acid sequence of the peptides has been determined. The largest labeled peptide was obtained with Lys-C protease and had the following sequence: -Ala-Phe-Val-Thr-Asp-Ile-Gly- Gly-Arg-Thr-Ser-His*-Ser-Ala-Ile-Met-Ala-Arg-Ser-Leu-Glu-Ile-Pro-Ala- Ile-Val-Gly-Thr-Lys-. It has previously been shown that the phosphoryl group is bound to the N-3 position of a histidyl residue in phosphorylated enzyme I. The single His in position 12 of the above peptide must therefore carry the phosphoryl group.     

Design of an Affinity Matrix for Purification of the Histamine H1 Receptor from Guinea Pig Cerebellum

H1 receptors from guinea pig cerebellum were solubilized using digitonin, and [125I]iodobolpyramine was used as a probe. [125I]Iodobolpyramine binding to this solubilized preparation occurred with a KD of 0.1 nM and a Bmax of 220 fmol/mg of protein and was inhibited by various H1 ligands with the expected potencies. Using a gel filtration procedure, a very sensitive radioassay was set up for detecting H1 activity in the solubilized preparation: 0.1 nM [125I]iodobolpyramine specific binding represented greater than 90% of total binding. Moreover, the synthesis is described of potent H1 antagonists that are mepyramine derivatives with an amino alkyl acylamido alkyl spacer arm. One of them, UCL 1057 (Ki = 0.5 nM), has been coupled to a Sepharose epoxy-activated resin. The resulting affinity matrix adsorbed selectively [125I]iodobolpyramine binding sites from the guinea pig cerebellum soluble preparation. In contrast, a Sepharose-glycine matrix was not able to adsorb these sites.     

Infrared spectra of the Gramicidin A transmembrane channel: the single-stranded-beta 6-helix

IR spectra are reported for preparations of Gramicidin A and malonyl Gramicidin A incorporated as the channel state in phospholipid structures. In this preparation Gramicidin A has already been shown to be unequivocally in the single-stranded beta-helical conformation. The result is an amide I frequency of 1633 +/- 1 cm-1. This demonstrates that the single-stranded beta-helix has an amide I frequency that has previously been considered to be diagnostic of antiparallel double-stranded beta-helix and of beta-sheet structures.     

Mode of Action of Pesticin

The mode of action of pesticin, a bacteriocin produced by many strains of Pasturella pestis, was studied. Pesticin action on macromolecular synthesis of a sensitive strain of Escherichia coli, strain , was found to have features similar to those of colicin E2-317 acting on the same strain. After exposure to pesticin, deoxyribonucleic acid synthesis was arrested and ribonucleic acid was degraded, but little effect was observed on protein synthesis. Pesticin, like colicin E2-317, induced lysogenic E. coli (P1), but, unlike the colicin, was active in the presence of dinitrophenol. Trypsin was found to reverse pesticin action up to 15 min after its addition at 40 C to E. coli . Pesticin action was studied on three sensitive bacterial strains, P. pestis 2C, P. pseudotuberculosis, and E. coli strain , which vary widely in their optimal growth temperature. P. pestis grows best at 29 C, P. pseudotuberculosis at 37 C, and E. coli at 40 C. It was found that pesticin action on all three strains was optimal at 40 C. Whereas the titer of pesticin was the same on all three strains when determined on agar, E. coli was the most sensitive to pesticin action in broth. No action of pesticin in broth on P. pseudotuberculosis was observed unless Ca ions were added. The effect was not immediate; that is, the cells had to be grown in a medium containing Ca⁺⁺ before they displayed sensitivity to pesticin.     

The role of tetrahydrobiopterin in the regulation of neuronal nitric-oxide synthase-generated superoxide

Tetrahydrobiopterin (H(4)B) is a critical element in the nitric-oxide synthase (NOS) metabolism of l-arginine to l-citrulline and NO(.). It has been hypothesized that in the absence of or under nonsaturating levels of L-arginine where O(2) reduction is the primary outcome of NOS activation, H(4)B promotes the generation of H(2)O(2) at the expense of O(2)(-.). The experiments were designed to test this hypothesis. To test this theory, two different enzyme preparations, H(4)B-bound NOS I and H(4)B-free NOS I, were used. Initial rates of NADPH turnover and O(2) utilization were found to be considerably greater in the H(4)B-bound NOS I preparation than in the H(4)B-free NOS I preparation. In contrast, the initial generation of O(2)(-.) from the H(4)B-free NOS I preparation was found to be substantially greater than that measured using the H(4)B-bound NOS I preparation. Finally, by spin trapping nearly all of the NOS I produced O(2)(-.), we found that the initial rate of H(2)O(2) production by H(4)B-bound NOS I was considerably greater than that for H(4)B-free NOS I.     

Antenna function of a chlorophyll ab protein complex of Photosystem I

The functional role of a chlorophyll $\text{a}\text{b}$ complex associated with Photosystem I (PS I) has been studied. The rate constant for P-700 photooxidation, K_P-700, which under light-limiting conditions is directly proportional to the size of the functional light-harvesting antenna, has been measured in two PS I preparations, one of which contains the chlorophyll $\text{a}\text{b}$ complex and the other lacking the complex. K_P-700 for the former preparation is half of that of the preparation which has the chlorophyll $\text{a}\text{b}$ complex present. This difference reflects a decrease in the functional light-harvesting antenna in the PS I complex devoid of the chlorophyll $\text{a}\text{b}$ complex. Experiments involving reconstitution of the chlorophyll $\text{a}\text{b}$ complex with the antenna-depleted PS I preparation indicate a substantial recovery of the K_P-700 rate. These results demonstrate that the chlorophyll $\text{a}\text{b}$ complex functions as a light-harvesting antenna in PS I.     

Effect of interferon type I on the in vivo persistence of Salmonella typhimurium

The influence of type I interferon on the persistence of S. typhimurium in the body of mice has been studied. The injection of the preparation of interferon has been shown to be conductive to the survival of the animals and to reduce the time of Salmonella persistence in the body. The injection of interferon enhances the phagocytic activity of macrophages in the peritoneal exudate of mice.     

Preparation of chloroplast DNA from pea plastids isolated in a medium of high ionic strength

A simple, rapid, and inexpensive method for the preparation and purification of chloroplast DNA (cpDNA) from pea has been developed. The crucial step is the isolation of chloroplasts in a medium of high ionic strength (I congruent equal to 1.40 M). CpDNA from pea prepared according to this method has successfully been used for restriction enzyme mapping, Southern transfers, and cloning.     

Photosystem I reaction centers fromChlamydomonas and higher plant chloroplasts

A photosystem I reaction center has been isolated fromChlamydomonas chloroplasts and compared with the photosystem I reaction center from higher plants. While the higher plant reaction center is active in cytochrome 552 photooxidation, theChlamydomonas preparation was not active unless salts were included in the assay medium or the pH was lowered to 5. Subunit III-depleted photosystem I reaction center from higher plants is also inactive in cytochrome 552 photooxidation in the absence of salts. As with theChlamydomonas reaction center, salts induced its activity. Subunit I of the photosystem I reaction center has tentatively been identified as the binding site of cytochrome 552.     

Determination of a new antibacterial agent (Ro 23-9424) by multidimensional high-performance liquid chromatography with ultraviolet detection and direct plasma injection

Analysis of a new antibacterial agent, Ro 23-9424 (I), in plasma has been complicated by the fact that its metabolite, fleroxacin (II), is formed not only in vivo, but also nonenzymatically by the hydrolysis of the ester bond of I. In order to minimize sample preparation time and possible hydrolysis during sample preparation, a high-performance liquid chromatographic procedure was developed which features direct injection of plasma and multidimensional chromatography. The first dimension size-exclusion separation allows plasma proteins to elute with the column void volume. The second dimension reversed-phase column provides a high-resolution separation dependent upon the hydrophobicity of the sample species. With a 5-μl injection, the limit of quantitation of the method is 0.35 μg/ml for I and 0.27 μg/ml for II. The method was used to determine steady state plasma vs. time profiles for I and II from 750 mg i.v. doses of I administered twice daily.     

Synthesis and biological evaluation of new camptothecin derivatives obtained by modification of position 20

The preparation and biological evaluation of a novel series of dimeric camptothecin derivatives are described. All the new compounds showed a significant ability to inhibit human tumor cell growth with IC(50) values ranging from 0.03 to 12.2 μM. The interference with the activity of the nuclear enzymes topoisomerases has been demonstrated, highlighting the poison effect of one of the obtained byproducts toward topoisomerase I. A moderate antiangiogenic activity has been demonstrated for one of the obtained compounds. Moreover, the effects of four new compounds on caspases activity and ROS generation have been studied on transgenic mouse cell.