Antimicrobial and antiviral activities of an actinomycete (Streptomyces sp.) isolated from a Brazilian tropical forest soil

A promising producer of bioactive compounds isolated from a Brazilian tropical soil was tested for its range of antimicrobial activities. Strain 606, classified as Streptomyces sp., could not be identified up to species level, suggesting a possible new taxon. The supernatant and 10 extracts and fractions, obtained by extraction and chromatographic techniques, presented antimicrobial activity using antibiograms. The methanolic fraction was highly active against pathogenic bacteria, phytopathogenic fungi and the human pathogenic yeast Candida albicans. It also possessed high antiviral activity inhibiting the propagation of an acyclovir-resistant herpes simplex virus type 1 strain on HEp-2 cells at non-cytotoxic concentration. The strong cytotoxic effect suggests an antitumour action. This revised version was published online in August 2006 with corrections to the Cover Date.     

Glycosylation of the phosphate binding protein, PstS, in Streptomyces coelicolor by a pathway that resembles protein O-mannosylation in eukaryotes

Previously mutations in a putative protein O -mannosyltransferase (SCO3154, Pmt) and a polyprenol phosphate mannose synthase (SCO1423, Ppm1) were found to cause resistance to phage, φC31, in the antibiotic producing bacteria Streptomyces coelicolor A3(2). It was proposed that these two enzymes were part of a protein O-glycosylation pathway that was necessary for synthesis of the phage receptor. Here we provide the evidence that Pmt and Ppm1 are indeed both required for protein O-glycosylation. The phosphate binding protein PstS was found to be glycosylated with a trihexose in the S. coelicolor parent strain, J1929, but not in the pmt ⁻ derivative, DT1025. Ppm1 was necessary for the transfer of mannose to endogenous polyprenol phosphate in membrane preparations of S. coelicolor . A mutation in ppm1 that conferred an E218V substitution in Ppm1 abolished mannose transfer and glycosylation of PstS. Mass spectrometry analysis of extracted lipids showed the presence of a glycosylated polyprenol phosphate (PP) containing nine repeated isoprenyl units (C₄₅-PP). S. coelicolor membranes were also able to catalyse the transfer of mannose to peptides derived from PstS, indicating that these could be targets for Pmt in vivo .     

Spontaneous Aggregation and Cytotoxicity of the β-Amyloid Aβ1–40: A Kinetic Model

The time dependency of the spontaneous aggregation of the fibrillogenic β-Amyloid peptide, Aβ^1–40, was measured by turbidity, circular dichroism, HPLC, and fluorescence polarization. The results by all methods were comparable and they were most consistent with a kinetic model where the peptide first slowly forms an activated monomeric derivative (AM), which is the only species able to initiate, by tetramerization, the formation of linear aggregates. The anti-Aβ antibody 6E10, raised against residues 1–17, at concentrations of 200–300 nM delayed significantly the aggregation of 50 μM amyloid peptide. The anti–Aβ antibody 4G8, raised against residues 17–24, was much less active in that respect, while the antibody A162, raised against the C-terminal residues 39–43 of the full-length Aβ was totally inactive at those concentrations. Concomitant with the aggregation experiments, we also measured the time dependency of the Aβ^1–40–induced toxicity toward SH-EP1 cells and hippocampal neurons, evaluated by SYTOX Green fluorescence, lactate dehydrogenase release, and activation of caspases. The extent of cell damage measured by all methods reached a maximum at the same time and this maximum coincided with that of the concentration of AM. According to the kinetic scheme, the latter is the only transient peptide species whose concentration passes through a maximum. Thus, it appears that the toxic species of Aβ^1–40 is most likely the same transient activated monomer that is responsible for the nucleation of fibril formation. These conclusions should provide a structural basis for understanding the toxicity of Aβ^1–40 in vitro and possibly in vivo.     

Assay of zofenopril and its active metabolite zofenoprilat by liquid chromatography coupled with tandem mass spectrometry

Zofenopril is a pro-drug designed to undergo metabolic hydrolysis yielding the active free sulfhydryl compound zofenoprilat, which is an angiotensin converting enzyme (ACE) inhibitor, endowed also with a marked cardioprotective activity. A simple, highly sensitive specific LC–MS–MS method was developed for the determination of zofenopril and zofenoprilat in human plasma. In order to prevent oxidative degradation of zofenoprilat and its internal standard, their free sulfhydryl groups were protected by treatment with N-ethylmaleimide (NEM), which produced the succinimide derivatives. The compounds and their corresponding fluorine derivatives, used as internal standards, were extracted from plasma with toluene. The reconstituted dried extracts were chromatographed and then monitored by a triple-stage-quadrupole instrument operating in the negative ion spray ionization mode. The method was validated over the concentration range of 1–300 ng/ml for zofenopril and 2–600 ng/ml for zofenoprilat. Inter- and intra-assay precision and accuracy of both zofenopril and zofenoprilat were better than 10%. The limit of quantitation was 1 ng/ml with zofenopril and 2 ng/ml with zofenoprilat. Extraction recovery proved to be on average 84.8% with zofenopril and 70.1% with zofenoprilat. Similar recoveries were shown by the above two internal standards. The method was applied to measure plasma concentrations of zofenopril and zofenoprilat in 18 healthy volunteers treated orally with zofenopril calcium salt at the dose of 60 mg.     

Nutrient and chlorophyll a temporal patterns in eutrophic mountain ponds with contrasting macrophyte coverage

Hydrobiologia - In shallow lakes, macrophytes have important effects on food webs, community structure and nutrient dynamics. For this reason they play a significant role in the restoration of...     

Horseradish peroxidase: a reliable or a misleading tool for the investigations on the origin of the proteins of the aqueous humor?

The concept of the blood-aqueous barrier is largely based on the use of horseradish peroxidase (HRP). The present investigation was designed to check its reliability as a macromolecular tracer, especially with regard to the transport of plasma proteins. Rabbits were killed 5 min to 24 h after being intravenously injected with HRP. The tracer diffused rapidly, reaching the aqueous humor of the eye in 3 min or less and was detected at high concentration in the narrow space between the outer epithelial layer of the ciliary epithelium and the wall of the pervious capillaries in the stroma of the processes. HRP appeared to migrate from the blood to the posterior chamber, permeating the tight junctions, viz., the anatomical basis of the blood-aqueous barrier. It was detected at higher concentration at the anterior surface of the iris, at short time intervals; this was interpreted as penetration of the tracer from the aqueous humor of the anterior chamber. The choroid was also labeled in continuation with the reaction in the stroma of the pars plana of the ciliary body which, in turn, sometimes reached the iris root. Therefore, the pervious blood vessels of the choroid could be a source of macromolecules for the iris root. HRP also induced the formation of lysosomes in the ciliary epithelium. This can hardly be accepted as the way in which plasma proteins are physiologically transported to the aqueous humor. However, the pathway of HRP migration over short time intervals seems to be in agreement with previous research indicating that the entrance of serum albumin into the posterior chamber is the first step of its incorporation into the aqueous humor. Received: 7 June 1996 / Accepted: 15 January 1997     

Localization of Endo-Oligopeptidase (EC 3.4.22.19) in the Rat Nervous Tissue

The subcellular and regional distribution of endo-oligopeptidase (EC 3.4.22.19), an enzyme capable of generating enkephalin by single cleavage from enkephalin-containing peptides, was determined by an enzymatic assay using metorphamide and by immunochemical techniques in the CNS of the rat. The rat CNS contains a membrane-associated form of endo-oligopeptidase, an enzyme predominantly associated with the soluble fraction of brain homogenates. Subcellular fractionation showed that approximately 17% of the total activity of the enzyme is associated with membrane fractions including synaptosomes. Synaptosomal membranes were prepared from neocortex, striatum, hypothalamus, medulla, spinal cord, and cerebellum. The amount of EC 3.4.22.19 activity solubilized by 3-[( 3-cholamidopropyl]dimethylammonio)-1-propanesulfonate from synaptosomal membranes was similar in neocortex, striatum, and hypothalamus, being three- to 10-fold greater than in spinal cord, cerebellum, and medulla. A polyclonal antibody exhibiting high affinity for endo-oligopeptidase was raised in rabbits against the purified rat brain enzyme and used to localize endo-oligopeptidase by Western blotting and by immunoperoxidase techniques. A strong band corresponding to the Mr of EC 3.4.22.19 was found in solubilized proteins obtained from synaptosomal membranes prepared from hypothalamus, neocortex, and striatum when subjected to Western blotting. The immunohistochemical localization of endo-oligopeptidase indicated that the immunoreactivity was confined to gray matter in regions known to be rich in peptide-containing neurons such as the striatum. In the cerebellum, a region poor in peptides, no staining could be detected. The nonuniform distribution of endo-oligopeptidase in rat brain suggests a role in neurotransmitter processing in the CNS.     

Staining of Oligodendroglia with Silver Ammino Tungstate in Unembedded and Embedded Material

Specimens of brain or spinal cord fixed in formalin, Cajal's formol-bromide, or Koenig, Groat and Windle's formalin-acacia can be used to stain oligodendrocytes in frozen, in paraffin, or in celloidin sections. The sections are soaked 3-5 min in 0.02% acetic acid, pH 3.4, then rinsed 2-3 sec in 3% H₂O₂ and transferred to a silver bath prepared as follows: Mix equal parts of 10% AgNO₃ and 10% Na₂WO₄, and dissolve the precipitate with concentrated NH₄OH; avoid an excess of ammonia. Silver at room temperature for 15-20 sec, develop in 1% formalin, dehydrate, and mount. For embedded material, prepare a mixture consisting of 1 part of 10% aqueous Aerosol MA and 4 parts of 10% Aerosol OT in 95% alcohol. Add 5 drops of this mixture to each 50 ml of dilute acetic acid and 3% H₂O₂; 5 drops to each 20 ml of the silver bath.