New insight into anodization of aluminium with focused ion beam pre-patterning.

The self-ordered anodic aluminium oxide (AAO) structure consists of micron-scale domains-defect-free areas with a hexagonal arrangement of pores. A substantial increase in domain size is possible solely by pre-patterning the aluminium surface in the form of a defect-free hexagonal array of concaves, which guide the pore growth during subsequent anodization. Among the numerous pre-patterning techniques, direct etching by focused gallium ion beam (Ga FIB) allows the preparation of AAO with a custom-made geometry through precise control of the irradiation positions, beam energy, and ion dosage. The main drawback of the FIB approach includes gallium contamination of the aluminium surface. Here, we propose a multi-step anodizing procedure to prevent gallium incorporation into the aluminium substrate. The suggested approach successfully covers a wide range of AAO interpore distances from 100 to 500 nm. In particular, anodization of FIB pre-patterned aluminium in 0.1 M phosphoric acid at 195 V to prepare AAO with the interpore distance of about 500 nm was demonstrated for the first time. The quantification of the degree of pore ordering reveals the fraction of pores in hexagonal coordination above 96% and the in-plane mosaicity below 3° over an area of about 1000µm2. Large-scale defect-free AAO structures are promising for creating photonic crystals and hyperbolic metamaterials with distinct functional properties.

Flexible enzymatic activation of artificial polyketide extender units by Streptomyces cinnamonensis into the monensin biosynthetic pathway.

Streptomyces cinnamonensis A495 is a variant of the monensin producer which instead of the native polyether antibiotic gives rise to antibiotic and anti-tumour shunt-product premonensin. Through the supplementation of the fermentation medium with suitable precursors, premonensin can be derivatized via the incorporation of new-to-nature extender units into the biosynthetic machinery. Polyketide extender units require activation, typically in form of coenzyme A-thioesters. These are membrane impermeable and thus in the past an artificial mimic was employed. Here, we show the use and preliminary characterization of a highly substrate promiscuous new enzyme for the endogenous thioester formation in a Streptomyces strain. These intracellularly activated alternative extender units are significantly better incorporated into premonensin than the synthetically activated counterparts. SIGNIFICANCE AND IMPACT OF THE STUDY: Polyketide natural products are of enormous relevance in medicine. The hit-rate in finding active compounds for the potential treatment of various diseases among this substance family of microbial origin is high. However, most polyketides require derivatization to render them suitable for the application. Of relevance in this field is the incorporation of artificial substances into the biogenesis of polyketides, hampered by both the microbial metabolism and the complexity of the enzymes involved. This manuscript describes the straightforward and selective biosynthetic incorporation of synthetic substances into a reduced polyketide and showcases a promising new enzyme to aid this purpose.

Colistin exposure as a risk factor for infections caused by inherently colistin resistant Enterobacteriaceae-a case-control study.

OBJECTIVES: Epidemiological studies have suggested an association between increased colistin use and selection for inherently colistin-resistant Enterobacteriaceae (ICRE). We aimed to evaluate whether colistin exposure is a risk factor for ICRE infection. METHODS: A matched 1:1 case-control study including patients recently hospitalized for ≥14 days with ICRE infection as cases matched with similar patients with a clinical isolate of a colistin-susceptible Enterobacteriaceae as controls was performed. Univariate analysis using McNemar test and multivariate analysis were conducted to explore risk factors for ICRE isolation, including colistin exposure 90 days before the positive culture. RESULTS: We included 446 patients, 223 cases and 223 controls matched for gender, age, department, year, source of culture and duration of hospitalization before positive culture. Colistin exposure was significantly associated with ICRE isolation in both univariate (14/223, 6.3% of cases versus 4/223, 1.8% of controls, p 0.031) and multivariate analyses (odds ratio 4.415, 95% CI 1.078-18.082). Curtailed functional capacity was a significant risk factor for ICRE as well. Exposure to other broad-spectrum antibiotics was associated with isolation of a colistin-susceptible pathogen. CONCLUSIONS: Exposure to colistin is associated with an increased risk of isolating an inherently colistin-resistant Enterobacteriaceae in patients with prolonged hospitalization. This should be taken into account while considering empirical therapy for such patients. Use of colistin should be judicious. The correlation between duration and magnitude of exposure and ICRE infection should be investigated in further studies.

Biosynthesis-driven structure-activity relationship study of premonensin-derivatives.

The controlled derivatization of natural products is of great importance for their use in drug discovery. The ideally rapid generation of compound libraries for structure-activity relationship studies is of particular concern. We here use modified biosynthesis for the generation of such a library of reduced polyketides to interfere with the oncogenic KRas pathway. The polyketide is derivatized via side chain alteration, and variations in its redox pattern and in its backbone chain length through manipulation in the corresponding polyketide synthase. Structural and biophysical analyses revealed the nature of the interaction between the polyketides and KRas-interacting protein PDE6δ. Non-natural polyketides with low nanomolar affinity to PDE6δ were identified.

Comparative effectiveness of a fixed-dose combination of losartan + HCTZ versus bisoprolol + HCTZ in patients with moderate-to-severe hypertension: results of the 6-month ELIZA trial.

BACKGROUND: The aim of this study was to compare the antihypertensive efficacy of losartan 100 mg + hydrochlorothiazide (HCTZ) 25 mg versus bisoprolol 10 mg + HCTZ 25 mg and their influence on arterial stiffness and central blood pressure (BP). METHODS: Of 60 patients with a mean BP of 173.3 ± 1.7/98.4 ± 1.2 mmHg, 59 were randomized to losartan + HCTZ (n = 32) or bisoprolol + HCTZ (n = 27). Amlodipine was added if target BP was not achieved at 1 month, and doxazosin was added if target BP was not achieved after 3 months. Body mass index, office and 24-hour ambulatory BP, pulse wave velocity (carotid-femoral [PWVE] and radial [PWVM]), noninvasive central systolic BP, augmentation index (AIx), laboratory investigations, and electrocardiography were done at baseline and after 6 months of treatment. RESULTS: Losartan + HCTZ was as effective as bisoprolol + HCTZ, with target office BP achieved in 96.9% and 92.6% of patients and target 24-hour BP in 75% and 66.7% of patients, respectively, after 6 months. Effective treatment of BP led to significant lowering of central systolic BP, but this was decreased to a significantly (P < 0.05) greater extent by losartan + HCTZ (-23.0 ± 2.3 mmHg) than by bisoprolol + HCTZ (-15.4 ± 2.9 mmHg) despite equal lowering of brachial BP. Factors correlated with central systolic BP and its lowering differed between the treatment groups. Losartan + HCTZ did not alter arterial stiffness patterns significantly, but bisoprolol + HCTZ significantly increased AIx. We noted differences in ΔPWVE, ΔPWVM, and ΔAIx between the groups in favor of losartan + HCTZ. Decreased heart rate was associated with higher central systolic BP and AIx in the bisoprolol + HCTZ group, but was not associated with increased AIx in the losartan + HCTZ group. CONCLUSION: Although both treatments decreased both office and 24-hour BP, losartan + HCTZ significantly decreased central systolic BP and had a more positive influence on pulse wave velocity, with a less negative effect of decreased heart rate on AIx and central systolic BP.

[Risk and prevention of teeth fluorosis in infants by feeding pattern changes].

Effect of fluorides in drinking water on fluorine content in breast milk, the food for infants of the 1-sty year of life, was investigated. On determining fluorine concentration in urine and its excretion, fluorine intake by the infants was calculated under various alternatives (breast, mixed and artificial) of feeding. It has been found the in mixed and especially in artificial feeding, fluorine intake by the infants acquires uncontrollable character exceeding several times the dose of fluorine intake with breast milk under natural feeding. That was predominantly fluorine of drinking water. Mathematical formula for calculation of fluorine content in the food cooked for feeding of infants was elaborated. A computer program was formed be means of which calculation, control and management of fluorine intake are carried out in feeding alteration.

[Problems of fluoride dosing to infants for dental fluorosis prevention].

Fluoride content in drinking water, breast milk, cow milk, additional food for newborns prepared with water containing different amount of fluoride was determined. Fluoride excretion in urine since the first days of birth and up to 4 months of postnatal development was investigated in breast and artificially fed infants. When a neonate was fed with breast milk, fluoride was received in the amount no more than 20 mkg/day. The additional food contained fluoride which water mainly had. Water with high level of fluorine increased its content in the additional food up to the values not comparable to those in breast milk that presented danger of dental fluorosis development. Data on fluorine content in drinking water were absolutely necessary to calculate daily fluorides consumption by infants and to prevent dental fluorosis.

DNA-binding activity of LndI protein and temporal expression of the gene that upregulates landomycin E production in Streptomyces globisporus 1912.

The gene lndI is involved in the pathway-specific positive regulation of biosynthesis of the antitumour polyketide landomycin E in Streptomyces globisporus 1912. LndI was overexpressed in Escherichia coli as a protein C-terminally fused to the intein-chitin-binding-domain tag and purified in a one-step column procedure. Results of in vivo LndI titration, DNA gel mobility-shift assays and promoter-probing experiments indicate that LndI is an autoregulatory DNA-binding protein that binds to its own gene promoter and to the promoter of the structural gene lndE. Enhanced green fluorescent protein was used as a reporter to study the temporal and spatial pattern of lndI transcription. Expression of lndI started before cells entered mid-exponential phase and peak expression coincided with maximal accumulation of landomycin E and biomass. In solid-phase analysis, lndI expression was evident in substrate mycelia but was absent from aerial hyphae and spores.

[Biosynthesis of hydrolytic enzymes during cocultivation of macro- and micromycetes]. / Biosintez gidroliticheskikh fermentov pri sovmestnom kul'tivirovanii makro- i mikromitsetov.

Effects of co-cultivation of higher Basidiomycetes and Phycomycetes on the biosynthesis of cellulases, amylases, and proteases was studied. Four optimal pairs of fungal cultures were selected. Of these, three pairs belonged to higher fungi, and one pair was constituted by fungi of distinct ecological groups, a macromycete and a micromycete. The activities of amylase and protease were 1.5 to 2 times higher, and the activity of cellulase was lower during the growth of higher fungi associations. The mixed association of the macromycete Schizophyllum commune and the micromycete Mucor sp. was the most active producer of hydrolytic enzymes. During the growth of this mixed association, a fourfold and 1.5-fold increases were observed in the activity of endoglucanase and protease, respectively, paralleled by stimulation of amylase formation.

N-terminal domains of plant poly(ADP-ribose) polymerases define their association with mitotic chromosomes.

Poly(ADP-ribos)ylation is a reversible protein modification that in higher plants is catalyzed by two structurally different poly(ADP-ribose) polymerases, App and Zap. In vivo imaging of green-fluorescent protein (GPF) fusions showed that both Zap and App were associated with chromatin through the cell cycle progression. The in vivo behaviour of the App-GFP protein fusions can be attributed to the activity of two NASA motifs that mediate protein-protein interactions and nucleic acid binding. Expression of Zap deletion variants revealed that both Zn fingers and helix-turn-helix domains contributed to the association with chromosomes, whereas the localization in the nucleoplasm was mostly determined by the Zn fingers. The results highlight novel properties of protein sequences found in plant poly(ADP-ribose) polymerases and suggest important functions for this class of nuclear enzymes in chromosome dynamics.

Arabidopsis coactivator ALY-like proteins, DIP1 and DIP2, interact physically with the DNA-binding domain of the Zn-finger poly(ADP-ribose) polymerase.

Two novel homologous Arabidopsis proteins, DIP1 and DIP2, interact with the DNA-binding domain of plant poly(ADP-ribose) polymerase (PARP) in the yeast two-hybrid system and in vitro. Their homology to the transcriptional coactivator ALY suggests that plant PARP may play a role in the regulation of transcription.

A mutation of the mitochondrial ABC transporter Sta1 leads to dwarfism and chlorosis in the Arabidopsis mutant starik.

A mutation in the Arabidopsis gene STARIK leads to dwarfism and chlorosis of plants with an altered morphology of leaf and cell nuclei. We show that the STARIK gene encodes the mitochondrial ABC transporter Sta1 that belongs to a subfamily of Arabidopsis half-ABC transporters. The severity of the starik phenotype is suppressed by the ectopic expression of the STA2 homolog; thus, Sta1 function is partially redundant. Sta1 supports the maturation of cytosolic Fe/S protein in Deltaatm1 yeast, substituting for the ABC transporter Atm1p. Similar to Atm1p-deficient yeast, mitochondria of the starik mutant accumulated more nonheme, nonprotein iron than did wild-type organelles. We further show that plant mitochondria contain a putative l-cysteine desulfurase. Taken together, our results suggest that plant mitochondria possess an evolutionarily conserved Fe/S cluster biosynthesis pathway, which is linked to the intracellular iron homeostasis by the function of Atm1p-like ABC transporters.

CEO1, a new protein from Arabidopsis thaliana, protects yeast against oxidative damage.

The Saccharomyces cerevisiae strain WYT, deficient in the YAP1 transcription factor, was used in a molecular screen to identify genes from Arabidopsis thaliana that could overcome the oxidative stress-sensitive phenotype of these yeast cells. A cDNA named CEO1 increased the tolerance to oxidative damage caused by tert-butylhydroperoxide of both the Yap1(-) mutant and the wild-type yeast. Additionally, in Yap1(-) yeast, CEO1 also induced cross-tolerance to oxidative damage caused by hydrogen peroxide and diamide. CEO1 was assigned as being part of a small gene family that, until now, is exclusively restricted to plants. In Arabidopsis, CEO1 was produced in all organs, especially in roots and stems. By using the yeast two-hybrid system, proteins that specifically interact with CEO1 in yeast were identified, and putative DNA-binding proteins were consistently recovered.

CEF, a sec24 homologue of Arabidopsis thaliana, enhances the survival of yeast under oxidative stress conditions.

Budding yeast strains that produced the Arabidopsis thaliana protein CEF or its amino-terminal proline-rich domain were more tolerant to hydroperoxides. CEF is homologous to animal and yeast Sec24 proteins. These data suggest that CEF plays a protective role through protein transport during growth under pro-oxidant conditions.

A novel NADPH:diamide oxidoreductase activity in arabidopsis thaliana P1 zeta-crystallin.

The zeta-crystallin (ZCr) gene P1 of Arabidopsis thaliana, known to confer tolerance toward the oxidizing drug 1,1'-azobis(N, N-dimethylformamide) (diamide) to yeast [Babiychuk, E., Kushnir, S., Belles-Boix, E., Van Montagu, M. & Inzé, D. (1995) J. Biol. Chem. 270, 26224], was expressed in Escherichia coli to characterize biochemical properties of the P1-zeta-crystallin (P1-ZCr). Recombinant P1-ZCr, a noncovalent dimer, showed NADPH:quinone oxidoreductase activity with specificity to quinones similar to that of guinea-pig ZCr. P1-ZCr also catalyzed the divalent reduction of diamide to 1,2-bis(N,N-dimethylcarbamoyl)hydrazine, with a kcat comparable with that for quinones. Two other azodicarbonyl compounds also served as substrates of P1-ZCr. Guinea-pig ZCr, however, did not catalyze the azodicarbonyl reduction. Hence, plant ZCr is distinct from mammalian ZCr, and can be referred to as NADPH:azodicarbonyl/quinone reductase. The quinone-reducing reaction was accompanied by radical chain reactions to produce superoxide radicals, while the azodicarbonyl-reducing reaction was not. Specificity to NADPH, as judged by kcat/Km, was > 1000-fold higher than that to NADH both for quinones and diamide. N-Ethylmaleimide and p-chloromercuribenzoic acid inhibited both quinone-reducing and diamide-reducing activities. Both NADPH and NADP+ suppressed the inhibition, but NADH did not, suggesting that sulfhydryl groups reside in the binding site for the phosphate group on the adenosine moiety of NADPH. The diamide-reducing activity of P1-ZCr accounts for the tolerance of P1-overexpressing yeast to diamide. Other possible physiological functions of P1-ZCr in plants are discussed.

The heat-shock element is a functional component of the Arabidopsis APX1 gene promoter.

Ascorbate peroxidases are important enzymes that detoxify hydrogen peroxide within the cytosol and chloroplasts of plant cells. To better understand their role in oxidative stress tolerance, the transcriptional regulation of the apx1 gene from Arabidopsis was studied. The apx1 gene was expressed in all tested organs of Arabidopsis; mRNA levels were low in roots, leaves, and stems and high in flowers. Steady-state mRNA levels in leaves or cell suspensions increased after treatment with methyl viologen, ethephon, high temperature, and illumination of etiolated seedlings. A putative heat-shock cis element found in the apx1 promoter was shown to be recognized by the tomato (Lycopersicon esculentum) heat-shock factor in vitro and to be responsible for the in vivo heat-shock induction of the gene. The heat-shock cis element also contributed partially to the induction of the gene by oxidative stress. By using in vivo dimethyl sulfate footprinting, we showed that proteins interacted with a G/C-rich element found in the apx1 promoter.

Higher plants possess two structurally different poly(ADP-ribose) polymerases.

One of the immediate reactions of the mammalian cell to many environmental stresses is a massive synthesis of poly(ADP-ribose), catalyzed by poly(ADP-ribose) polymerase (PARP). Most of the biological functions attributed to PARP are inferred from experimentation with mammalian cells. In plants, the biology of PARP may be more complicated and diverse than was previously thought. Two poly(ADP-ribose) polymerase homologues were found in plants, the classical Zn-finger-containing polymerase (ZAP) and the structurally non-classical PARP proteins (APP and NAP), which lack the characteristic N-terminal Zn-finger domain. By enzymatic and cytological experiments the recombinant APP protein was shown to be located in the nucleus and to possess DNA-dependent poly(ADP-ribose) polymerase activity in yeast. The nuclear localization was further confirmed by the analysis of transgenic tobacco plants that expressed a translational gene fusion between APP and the bacterial beta-glucuronidase. The app promoter was transcriptionally up-regulated in cells pre-determined to die because of deficiency in a DNA ligase I.

Processing of a chimeric protein in chloroplasts is different in transgenic maize and tobacco plants.

Transgenic maize (Zea mays L.) and tobacco (Nicotiana tabacum Petit Havana SR1) plants have been generated, which overproduce a mitochondrial Nicotiana plumbaginifolia manganese superoxide dismutase (MnSOD) in chloroplasts. For this, the mature MnSOD-coding sequence was fused to a chloroplast transit peptide from a Pisum sativum ribulose-1,5-bisphosphate carboxylase (Rubisco) gene and expression of the chimeric gene was driven by the cauliflower mosaic virus (CaMV) 35S promoter. The transgenic MnSOD gene product was correctly targeted to the chloroplasts both in maize and tobacco. However, despite the use of the CaMV 35S promoter, the MnSOD was predominantly localized in the chloroplasts of the bundle sheath cells of maize. Furthermore, the transit peptide was cleaved off at a different position in maize and tobacco.

[The clinical characteristics of panic attacks in adolescents with the cardiac form of the autonomic dystonia syndrome]. / Klinicheskie osobennosti panicheskikh atak u podrostkov s kardial'noi formoi sindroma vegetativnoi distonii.

Peculiarities of paroxysmal course of autonomic dystonia of cardiac type were studied in 148 juveniles aged 14-15 years. It was found that frequency of panic attacks was very high, i.e. they occurred in 67-92.4% of the cases in dependence on severity of the disease. Panic attacks had no definite clinical picture at the onset. However, as the intensity of the main clinical manifestation of the disease (chest-pain syndrome) increased they acquired clear-cut sympatho-adrenal or vago-insular direction. Short duration and incomplete nels are typical for panic attacks in these patients.

Efficient gene tagging in Arabidopsis thaliana using a gene trap approach.

Large quantities of DNA sequence information about plant genes are rapidly accumulating in public databases, but to progress from DNA sequence to biological function a mutant allele for each of the genes ideally should be available. Here we describe a gene trap construct that allowed us to disrupt transcribed genes with a high efficiency in Arabidopsis thaliana. In the T-DNA vector used, the expression of a bacterial reporter gene coding for neomycin phosphotransferase II (nptII) depends on the in vivo generation of a translation fusion upon the T-DNA integration into the Arabidopsis genome. Analysis of 20 selected transgenic lines showed that 12 lines are T-DNA insertion mutants. The disrupted genes analyzed encoded ribosomal proteins (three lines), aspartate tRNA synthase, DNA ligase, basic-domain leucine zipper DNA binding protein, ATP-binding cassette transporter, and five proteins of unknown function. Four tagged genes were new for Arabidopsis. The results presented here suggest that gene trapping, using nptII as a reporter gene, can be as high as 80% and opens novel perspectives for systematic gene tagging in A. thaliana.