Reactions of plant copper/topaquinone amine oxidases with N6-aminoalkyl derivatives of adenine

Plant copper/topaquinone-containing amine oxidases (CAOs, EC 1.4.3.6) are enzymes oxidising various amines. Here we report a study on the reactions of CAOs from grass pea (Lathyrus sativus), lentil (Lens esculenta) and Euphorbia characias, a Mediterranean shrub, with N6-aminoalkyl adenines representing combined analogues of cytokinins and polyamines. The following compounds were synthesised: N6-(3-aminopropyl)adenine, N6-(4-aminobutyl)adenine, N6-(4-amino-trans-but-2-enyl) adenine, N6-(4-amino-cis-but-2-enyl) adenine and N6-(4-aminobut-2-ynyl) adenine. From these, N6-(4-aminobutyl) adenine and N6-(4-amino-trans-but-2-enyl)adenine were found to be substrates for all three enzymes (Km approximately 10(-4)M). Absorption spectroscopy demonstrated such an interaction with the cofactor topaquinone, which is typical for common diamine substrates. However, only the former compound provided a regular reaction stoichiometry. Anaerobic absorption spectra of N6-(3-aminopropyl)adenine, N6-(4-amino-cis-but-2-enyl)adenine and N6-(4-aminobut-2-ynyl)adenine reactions revealed a similar kind of initial interaction, although the compounds finally inhibited the enzymes. Kinetic measurements allowed the determination of both inhibition type and strength; N6-(3-aminopropyl)adenine and N6-(4-amino-cis-but-2-enyl)adenine produced reversible inhibition (Ki approximately 10(-5) - 10(-4) M) whereas, N6-(4-aminobut-2-ynyl)adenine could be considered a powerful inactivator.     

Cloning, Biochemical Properties, and Distribution of Mycobacterial Haloalkane Dehalogenases

Haloalkane dehalogenases are enzymes that catalyze the cleavage of the carbon-halogen bond by a hydrolytic mechanism. Genomes of Mycobacterium tuberculosis and M. bovis contain at least two open reading frames coding for the polypeptides showing a high sequence similarity with biochemically characterized haloalkane dehalogenases. We describe here the cloning of the haloalkane dehalogenase genes dmbA and dmbB from M.bovis 5033/66 and demonstrate the dehalogenase activity of their translation products. Both of these genes are widely distributed among species of the M. tuberculosis complex, including M. bovis, M. bovis BCG, M.africanum, M. caprae, M. microti, and M. pinnipedii, as shown by the PCR screening of 48 isolates from various hosts. DmbA and DmbB proteins were heterologously expressed in Escherichia coli and purified to homogeneity. The DmbB protein had to be expressed in a fusion with thioredoxin to obtain a soluble protein sample. The temperature optimum of DmbA and DmbB proteins determined with 1,2-dibromoethane is 45°C. The melting temperature assessed by circular dichroism spectroscopy of DmbA is 47°C and DmbB is 57°C. The pH optimum of DmbA depends on composition of a buffer with maximal activity at 9.0. DmbB had a single pH optimum at pH 6.5. Mycobacteria are currently the only genus known to carry more than one haloalkane dehalogenase gene, although putative haloalkane dehalogenases can be inferred in more then 20 different bacterial species by comparative genomics. The evolution and distribution of haloalkane dehalogenases among mycobacteria is discussed.     

In vivo photoprotection mechanisms observed from leaf spectral absorbance changes showing VIS–NIR slow-induced conformational pigment bed changes

Photosynthesis Research - Regulated heat dissipation under excessive light comprises a complexity of mechanisms, whereby the supramolecular light-harvesting pigment–protein complex (LHC)...     

Tissue reaction to three different types of tissue glues in an experimental aorta dissection model: a quantitative approach

Tissue glues are used during surgical treatment of acute aorta dissection although some glues release toxic products and thus alter the histological structure of the vessel wall. The aim of our study was to use a porcine experimental model of infrarenal aorta dissection to compare histological changes of the vessel wall 1, 6 and 12 months after application of BioGlue, Gelatin-resorcin-formaldehyde (GRF) glue and Tissucol. For quantification, stereological methods were used. All types of glue caused stenosis, GRF most and Tissucol least severely. With increasing postoperative survival time, stenosis was again reduced. Elastine length density decreased with increasing survival time in Control as well as in all Experimental groups. The immunohistochemical phenotype of vascular smooth muscle cells was similar in Tissucol and Control samples. In GRF samples, actin, desmin and vimentin expression changed most severely. Similarly, number and distribution of vasa vasorum in the aortic wall was altered most severely in GRF samples. They tended to return to normal with increasing postoperative survival time, but at a slow rate in the GRF samples. It can be concluded that GRF causes the most severe histopathological changes within the treated aorta, which could be a reason for late failures of dissection surgery. However, glue handling and adhesive properties have to be taken into account, too, when certain glue is chosen for surgical intervention. Increased inflammation and vascularisation might even stabilise the aortic wall. Long-term experimental studies would be helpful to assess healing processes after initial disorganisation of the aortic wall structure.     

High level expression of human enteropeptidase light chain in Pichia pastoris

Human enterokinase (enteropeptidase, rhEP), a serine protease expressed in the proximal part of the small intestine, converts the inactive form of trypsinogen to active trypsin by endoproteolytic cleavage. The high specificity of the target site makes enterokinase an ideal tool for cleaving fusion proteins at defined cleavage sites. The mature active enzyme is comprised of two disulfide-linked polypeptide chains. The heavy chain anchors the enzyme in the intestinal brush border membrane, whereas the light chain represents the catalytic enzyme subunit. The synthetic gene encoding human enteropeptidase light chain with His-tag added at the C-terminus to facilitate protein purification was cloned into Pichia pastoris expression plasmids under the control of an inducible AOX1 or constitutive promoters GAP and AAC. Cultivation media and conditions were optimized as well as isolation and purification of the target protein. Up to 4 mg/L of rhEP was obtained in shake-flask experiments and the expression level of about 60-70 mg/L was achieved when cultivating in lab-scale fermentors. The constitutively expressing strains proved more efficient and less labor-demanding than the inducible ones. The rhEP was immobilized on AV 100 sorbent (Iontosorb) to allow repeated use of enterokinase, showing specific activity of 4 U/mL of wet matrix.     

How to asses, visualize and compare the anisotropy of linear structures reconstructed from optical sections--a study based on histopathological quantification of human brain microvessels

Three-dimensional analyses of the spatial arrangement, spatial orientation and preferential directions of systems of fibers are frequent tasks in many scientific fields, including the textile industry, plant biology and tissue modeling. In biology, systems of oriented and branching lines are often used to represent the three-dimensional directionality and topology of microscopic blood vessels supplying various organs. In our study, we present a novel p(χ2) (chi-square) method for evaluating the anisotropy of line systems that involves comparing the observed length densities of lines with the discrete uniform distribution of an isotropic line system with the χ2-test. Using this method in our open source software, we determined the rose of directions, preferential directions and level of anisotropy of linear systems representing the microscopic blood vessels in samples of various regions from human brains (cortex, subcortical gray matter and white matter). The novel method was compared with two other methods used for anisotropy quantification (ellipsoidal and fractional anisotropy). All three methods detected different levels of anisotropy of blood microvessels in human brain. The microvascular bed in the cortex was closer to an isotropic network, while the microvessels supplying the white matter appeared to be an anisotropic and direction-sensitive system. All three methods were able to determine the differences between various brain regions. The advantage of our p(χ2) method is its high correlation with the number of preferential directions of the line system. However, the software, named esofspy, is able to calculate all three of the measures of anisotropy compared and documented in this paper, thus making the methods freely available to the scientific community.     

Phosphoinositide 3-kinase inhibition enables retinoic acid-induced neurogenesis in monolayer culture of embryonic stem cells

Retinoic acid (RA) is able to induce the differentiation of embryonic stem cells into neuronal lineages. The mechanism of this effect is unknown but it has been evidenced to be dependent on the formation of floating spheroids called embryoid bodies. Results presented here show that the inhibition of phosphoinositide 3-kinase signaling pre-determines mouse embryonic stem cells to RA induced neurogenesis in monolayer culture with no need of embryoid bodies formation.     

Non-Enzymatic Oligomerization of 3’, 5’ Cyclic AMP

Recent studies illustrate that short oligonucleotide sequences can be easily produced from nucleotide precursors in a template-free non-enzymatic way under dehydrating conditions, i.e. using essentially dry materials. Here we report that 3’,5’ cyclic AMP may also serve as a substrate of the reaction, which proceeds under moderate conditions yet with a lower efficiency than the previously reported oligomerization of 3’,5’ cyclic GMP. Optimally the oligomerization requires (i) a temperature of 80°C, (ii) a neutral to alkaline environment and (iii) a time on the order of weeks. Differences in the yield and required reaction conditions of the oligomerizations utilizing 3’,5’ cGMP and cAMP are discussed in terms of the crystal structures of the compounds. Polymerization of 3’,5’ cyclic nucleotides, whose paramount relevance in a prebiotic chemistry context has been widely accepted for decades, supports the possibility that the origin of extant genetic materials might have followed a direct uninterrupted path since its very beginning, starting from non-elaborately pre-activated monomer compounds and simple reactions.     

Increased urinary zinc excretion in cancer patients is linked to immune activation and renal tubular cell dysfunction

Urinary zinc excretion is known to be increased in cancer patients, but the pathogenesis of this phenomenon remains uncertain. Both skeletal muscle catabolism and renal tubular cell dysfunction have been proposed to explain this observation. We have investigated urinary zinc and N-acetyl--d-glucosaminidase (NAG), an indicator of renal tubular cell dysfunction, as well as serum neopterin, an index of systemic immune activation, in 22 patients with cancer and seven controls. Both serum neopterin and urinary zinc were significantly elevated in cancer patients (15.8 ± 12.7 versus 7.3 ± 2.3 nmol l^–1 and 1.77 ± 0.80 versus 1.21 ± 0.41 mmol mol^–1 creatinine, P < 0 and P < 0.05, respectively), while NAG was similar in cancer patients and the controls (13.58 ± 13.80 versus 13.68 ± 12.19 kat mol^–1 creatinine). A significant correlation was observed between serum neopterin and urine zinc (rs = 0.5119, P < 0.02), serum neopterin and urine NAG (rs = 0.6761, P < 0.002), and urinary zinc and NAG (rs = 0.6348, P < 0.002). In conclusion, the present data indicate a link between urinary zinc excretion and immune activation as well as renal tubular cell dysfunction. In addition, renal tubular cell dysfunction appears to be linked to immune activation.     

Expression of glycosylated haloalkane dehalogenase LinB in Pichia pastoris

Heterologous expression of the bacterial enzyme haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26 in methylotrophic yeast Pichia pastoris is reported. The haloalkane dehalogenase gene linB was subcloned into the pPICZalphaA vector and integrated into the genome of P. pastoris. The recombinant LinB secreted from the yeast was purified to homogeneity and biochemically characterized. The deglycosylation experiment and mass spectrometry measurements showed that the recombinant LinB expressed in P. pastoris is glycosylated with a 2.8 kDa size of high mannose core. The specific activity of the glycosylated LinB was 15.6 +/- 3.7 micromol/min/mg of protein with 1,2-dibromoethane and 1.86 +/- 0.36 micromol/min/mg of protein with 1-chlorobutane. Activity and solution structure of the protein produced in P. pastoris is comparable with that of recombinant LinB expressed in Escherichia coli. The melting temperature determined by the circular dichroism (41.7+/-0.3 degrees C for LinB expressed in P. pastoris and 41.8 +/- 0.3 degrees C expressed in E. coli) and thermal stability measured by specific activity to 1-chlorobutane were also similar for two enzymes. Our results show that LinB can be extracellularly expressed in eukaryotic cell and glycosylation had no effect on activity, protein fold and thermal stability of LinB.