Increased vascular permeability in the circumventricular organs of adult rat brain due to stimulation by extremely low frequency magnetic fields

It has been demonstrated that the exposure of biological systems to magnetic fields (MFs) can produce several beneficial effects: tissue recovery in chronic wounds, re‐establishment of blood circulation after tissue ischemia or in necrotic tissues, improvement after epileptic episodes, angiogenesis, etc. In the current study, the effects of extremely low frequency (ELF) MF on the capillaries of some circumventricular organs (CVOs) are demonstrated; a vasodilator effect is reported as well as an increase in their permeability to non‐liposoluble substances. For this study, 96 Wistar male rats (250 g body mass) were used and divided into three groups of 32 rats each: a control group (no treatment); a sham ELF‐MF group; and an experimental group subjected to ELF‐MF (120 Hz harmonic waves and 0.66 mT, root mean square) by the use of Helmholtz coils. All animals were administered colloidal carbon (CC) intravenously to study, through optical and transmission electron microscopy, the capillary permeability in CVOs and the blood–brain barrier (BBB) in brain areas. An increase in capillary permeability to CC was detected in the ELF‐MF‐exposed group as well as a significant increase in vascular area (capillary vasodilation); none of these effects were observed in individuals of the control and sham ELF‐MF groups. It is important to investigate the mechanisms involved in the phenomena reported here in order to explain the effects of ELF‐MF on brain vasculature. Bioelectromagnetics 34:145–155, 2013. © 2012 Wiley Periodicals, Inc.     

Inflammation stimulates hypoxia-inducible factor-1α regulatory activity in 3T3-L1 adipocytes with conditioned medium from lipopolysaccharide-activated RAW 264.7 macrophages

Obesity is a multifactorial, chronic, inflammatory disease that involves different processes, such as adipose tissue hypoxia. The aim of the current study was to characterize the effects of conditioned medium (CM) from lipopolysaccharide (LPS)-activated macrophages on the regulation of hypoxia-inducible factor 1α (HIF-1α)-related genes in murine adipocytes. For the in vitro analyses, 3T3-L1 murine adipocytes (9 days postdifferentiation) were incubated either in CM (25% medium of RAW 264.7 murine macrophages with 24 hr 500 ng/ml LPS), LPS at 500 ng/ml, or hypoxia (Hx; 1% O₂, 94% N₂, 5% CO₂) for 24 hr. For the in vivo experiments, mice were fed a high-fat diet. Both epididymal white adipose tissue (eWAT) and adipocytes in CM showed upregulation of Glut1, Mcp1, Il10, Tnf, and Il1b. The secretion of IL-6, TNF-α, and MCP-1 was also increased in CM-treated adipocytes. Moreover, increased levels of HIF-1α subunit and nuclear factor kappa B p65 were found after CM treatment, linking Hx, and inflammation. HIF-1α directly bound vascular endothelial growth factor A (Vegfa) and uncoupling protein 2 (Ucp2) genes, up- and downregulating its expression, respectively. Furthermore, the oxygen consumption rate was 30% lower in CM. The siRNA knockdown of mammalian target of rapamycin (Mtor) reversed the induction of HIF-1α found in CM. The macrophage infiltration simulated through CM seems to be a similar environment to an abnormally enlarged eWAT. We have evidenced that HIF-1α plays a regulatory role in the expression of Vegfa and Ucp2 in CM. Finally, the inhibition of the mTOR pathway prevented the HIF-1α activation induced by CM. The involvement of HIF-1α under proinflammatory conditions provides insight into the origins of Hx in obesity.     

Bioclimatology of beech (Fagus sylvatica L.) in the Eastern Alps: spatial and altitudinal climatic signals identified through a tree-ring network

Aim To identify the dominant spatial patterns of Fagus sylvatica radial growth in the Eastern Alps, and to understand their relationships to climate variation and bioclimatic gradients. Location Fourteen beech stands in the Eastern Alps, growing between 200 and 1500 m a.s.l. in Italy, Slovenia and Austria. Methods At each site, trees were sampled using increment borers or by taking discs from felled trees. Cores and discs were processed by measuring and crossdating ring width. Ring width series were standardized, averaged, and prewhitened to obtain site chronologies. Hierarchical Cluster Analysis (HCA) and Principal Components Analysis of prewhitened site chronologies were used to identify spatial and altitudinal growth patterns, related to the bioclimatic position of each stand. Bootstrap correlation and response functions were computed between monthly climatic variables and either principal component scores or composite chronologies from stands associated by HCA. The stability of dendroclimatic signals was analyzed by moving correlation functions (MCF). Correlation analysis (teleconnections) based on a data base of 37 Italian and Slovenian beech tree‐ring chronologies revealed the spatial extent of principal component scores. Results Sampled trees were 200–400 years old, representing the oldest beech trees that have been crossdated for the Alps to date. Maximum age was directly related to altitude and to the presence of historical forms of conservation. Tree‐ring parameters varied according to geographic patterns and the age of sampled trees. Stands were bioclimatically organized according to their location, and with reference to their elevation and distance from the Adriatic Sea. A direct response to winter temperature was found at all elevations. The altitudinal gradient ranged from low‐elevation stands, characterized by a Mediterranean‐type, late spring–summer drought signal, to mountain and high‐elevation stands, characterized by a direct response to growing season temperature plus an inverse response to the previous year’s July temperature. The mountain and high‐elevation signal was evident in Austria, the Central Alps and Slovenia, while the low‐elevation signal was confined to mountains adjacent to the Adriatic Sea. MCF revealed trends in the response to climatic factors affecting tree‐ring formation in mountain and high‐mountain stands linked to climatic warming. Main conclusions Dendroclimatic networks can be used for bioclimatic studies of tree populations. A biogeographical separation emerged between the Alps and the Apennines at the upper elevations, while different degrees of mediterraneity distinguished sites at lower elevations. This information will be useful in assessing any future climate‐related bioclimatic shifts, especially for forests at ecotones and along altitudinal gradients.     

Efficiency of insecticidal crystal protein production in a Bacillus thuringiensis mutant with derepressed expression of the terminal oxidase aa 3 during sporulation

A Bacillus thuringiensis respiratory mutant (AB1 strain) that shows premature sporulation and insecticidal crystal protein (ICP) production was isolated. The mutant strain harbours the cryIC and cryID insecticidal genes and could be important for the production of ICP highly toxic to Spodoptera sp. The mutant was selected by its increased capacity to oxidize. N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD). In this strain, cytochrome aa ₃ expression is not repressed during the sporulation phase, in contrast with the wild-type strain. The growth, spore production, dissolved O₂, O₂ consumption, CO₂ evolution rate and ICP production were recorded as a function of time. The AB1 mutant strain has a similar growth yield to the wild-type strain, but begins sporulation at least 4 h earlier. The AB1 strain consumes 74.5% more O₂ than the wild-type strain, during the fermentation process. The mutation on strain AB1 has an important positive effect on ICP production. This procedure shows that ICP production could be increased during fermentation by increasing the respiration capacity of Bacillus thuringiensis. Correspondence to: A. Bravo     

Determination of pseudouridine and other nucleosides in human blood serum by high-performance liquid chromatography

A specific, sensitive, and rapid method to measure pseudouridine in human blood serum is described. The method is based on the following steps: (i) deproteinization of serum samples by filtration on membrane cones or by acetonitrile; (ii) purification of nucleosides and concentration of the sample by affinity chromatography on phenylboronate gel followed by lyophilization; and (iii) separation of nucleosides and their quantitation by reserse-phase high-performance liquid chromatography.The pseudouridine mean value in 30 normal subjects was 2.52 ± 0.28 nmol/ml. The procedure also allows the identification of inosine, uridine, guanosine, and adenosine. Nevertheless, the presence in human blood serum of enzymatic activities which convert adenosine to inosine and cytidine to uridine prevents the precise quantitation of these nucleosides. All the compounds were identified by comparing their retention times and absorbance ratios ($\text{A}{}_{280}\text{A}{}_{254}$) with those of pure compounds, as well as by cochromatography.     

Mechanisms involved in the increased sensitivity ofEscherichia coli to microcin 15m at 42°C

Escherichia coli cells show a markedly increased sensitivity to the antibiotic microcin 15m when briefly treated at 42°C as compared to the effect at 37°C. Furthermore, mutants resistant to the microcin at 37°C become sensitive at 42°C at microcin concentrations that are inactive at 37°C. This effect can be overcome byl-methionine. The mechanism involved seems to be based on an apparent inactivation of the homoserine-O-transsuccinylase activity. As previously established, this enzyme suffers a reversible partial inactivation when the cells are shifted to 42°C and the action of the microcin at this temperature seems to bring this process to a virtually irreversible stage. In mixed cultures of the microcin-producing strain and oneE. coli strain sensitive to the antibiotic, a much stronger growth inhibition of the latter strain has been observed at 42°C than at 37°C.     

Analysis of the β-1,3-Glucanolytic System of the Biocontrol Agent Trichoderma harzianum

The biocontrol agent Trichoderma harzianum IMI206040 secretes β-1,3-glucanases in the presence of different glucose polymers and fungal cell walls. The level of β-1,3-glucanase activity secreted was found to be proportional to the amount of glucan present in the inducer. The fungus produces at least seven extracellular β-1,3-glucanases upon induction with laminarin, a soluble β-1,3-glucan. The molecular weights of five of these enzymes fall in the range from 60,000 to 80,000, and their pIs are 5.0 to 6.8. In addition, a 35-kDa protein with a pI of 5.5 and a 39-kDa protein are also secreted. Glucose appears to inhibit the formation of all of the inducible β-1,3-glucanases detected. A 77-kDa glucanase was partially purified from the laminarin culture filtrate. This enzyme is glycosylated and belongs to the exo-β-1,3-glucanase group. The properties of this complex group of enzymes suggest that the enzymes might play different roles in host cell wall lysis during mycoparasitism.     

Identification of Two Genes from Streptomyces argillaceus Encoding Glycosyltransferases Involved in Transfer of a Disaccharide during Biosynthesis of the Antitumor Drug Mithramycin

Mithramycin is an antitumor polyketide drug produced by Streptomyces argillaceus that contains two deoxysugar chains, a disaccharide consisting of two d-olivoses and a trisaccharide consisting of a d-olivose, a d-oliose, and a d-mycarose. From a cosmid clone (cosAR3) which confers resistance to mithramycin in streptomycetes, a 3-kb PstI-XhoI fragment was sequenced, and two divergent genes (mtmGI and mtmGII) were identified. Comparison of the deduced products of both genes with proteins in databases showed similarities with glycosyltransferases and glucuronosyltransferases from different sources, including several glycosyltransferases involved in sugar transfer during antibiotic biosynthesis. Both genes were independently inactivated by gene replacement, and the mutants generated (M3G1 and M3G2) did not produce mithramycin. High-performance liquid chromatography analysis of ethyl acetate extracts of culture supernatants of both mutants showed the presence of several peaks with the characteristic spectra of mithramycin biosynthetic intermediates. Four compounds were isolated from both mutants by preparative high-performance liquid chromatography, and their structures were elucidated by physicochemical methods. The structures of these compounds were identical in both mutants, and the compounds are suggested to be glycosylated intermediates of mithramycin biosynthesis with different numbers of sugar moieties attached to C-12a-O of a tetracyclic mithramycin precursor and to C-2-O of mithramycinone: three tetracyclic intermediates containing one sugar (premithramycin A1), two sugars (premithramycin A2), or three sugars (premithramycin A3) and one tricyclic intermediate containing a trisaccharide chain (premithramycin A4). It is proposed that the glycosyltransferases encoded by mtmGI and mtmGII are responsible for forming and transferring the disaccharide during mithramycin biosynthesis. From the structures of the new metabolites, a new biosynthetic sequence regarding late steps of mithramycin biosynthesis can be suggested, a sequence which includes glycosyl transfer steps prior to the final shaping of the aglycone moiety of mithramycin.

Many bioactive drugs contain sugars attached to their aglycones which are usually important or, in some cases, essential for bioactivity. Most of these sugars belong to the family of the 6-deoxyhexoses (6-DOH) (18, 20, 27) and are transferred to the different aglycones as late steps in biosynthesis. Genes involved in the biosynthesis of different 6-DOH have been reported elsewhere and participate in the biosynthesis of erythromycin (9, 12, 31, 38, 39), daunorubicin (13, 26, 36), mithramycin (22), granaticin (2), streptomycin (10, 28), and tylosin (14, 23). However, information about the glycosyltransferases (GTFs) responsible for the transfer of the sugars to the respective aglycones is quite scarce. So far, only two GTFs from antibiotic producers have been biochemically characterized in detail, and they are involved in macrolide inactivation: Mgt, from Streptomyces lividans, a nonmacrolide producer (7, 17); and OleD, from the oleandomycin producer Streptomyces antibioticus (15, 29), which inactivates oleandomycin by addition of glucose to the 2′-OH group of the desosamine attached to the macrolactone ring (40). In the last several years, a few genes have been proposed to encode GTFs involved in the transfer of sugars to various aglycones during biosynthesis: dnrS and dnrH, from Streptomyces peucetius, involved in daunorubicin (26) and baumycin (36) biosynthesis, respectively; gra-orf5, involved in granaticin biosynthesis (2); eryCIII and eryBV, involved in the transfer of desosamine and mycarose, respectively, in erythromycin biosynthesis (12, 32, 38); and tylM2, from Streptomyces fradiae, involved in sugar transfer during tylosin biosynthesis (14).Mithramycin (Fig. ​(Fig.1)1) is an aromatic polyketide which shows antibacterial activity against gram-positive bacteria and also antitumor activity (30, 37). Together with the chromomycins and the olivomycins, mithramycin constitutes the so-called aureolic acid group of antitumor drugs. The polyketide moiety of mithramycin is derived from the condensation of 10 acetate building blocks in a series of reactions catalyzed by a type II polyketide synthase (5, 21). The mithramycin aglycone is glycosylated at positions 6 and 2 with disaccharide (d-olivose- d-olivose) and trisaccharide (d-olivose-d-oliose-d-mycarose) moieties, respectively. All of these sugars belong to the 6-DOH family. In the mithramycin pathway, two genes (mtmD and mtmE) encoding two enzymes (glucose-1-phosphate:TTP thymidylyl transferase and dTDP-4,6-dehydratase, respectively) involved in the biosynthesis of the mithramycin 6-DOH have been cloned, and their participation in mithramycin biosynthesis has been demonstrated by insertional inactivation (22). Here we report the characterization of two Streptomyces argillaceus genes (mtmGI and mtmGII) that encode two putative GTFs responsible for the formation and transfer of the disaccharide chain. Inactivation of these genes by gene replacement showed identical accumulated compounds and allowed the isolation of four glycosylated compounds which are likely to be intermediates in mithramycin biosynthesis. Open in a separate windowFIG. 1Structures of mithramycin, premithramycinone, and the new premithramycins.