Detection of cytokinins and auxin in plant tissues using histochemistry and immunocytochemistry

We report a new method for histochemical localization of cytokinins (CKs) in plant tissues based on bromophenol blue/silver nitrate staining. The method was validated by immunohistochemistry using anti-trans-zeatin riboside antibody. Indole-3-acetic acid (auxin, IAA) was localized by anti-IAA antibody in plant tissues as a proof for IAA histolocalization. We used root sections, because they are major sites of CKs synthesis, and insect galls of Piptadenia gonoacantha that accumulate IAA. Immunostaining confirmed the presence of zeatin and sites of accumulation of IAA indicated by histochemistry. The colors developed by histochemical reactions in free-hand sections of plant tissues were similar to those obtained by thin layer chromatography (TLC), which reinforced the reactive sites of zeatin. The histochemical method for detecting CKs is useful for galls and roots, whereas IAA detection is more efficient for gall tissues. Therefore, galls constitute a useful model for validating histochemical techniques due to their rapid cell cycles and relatively high accumulation of plant hormones.     

Isolation and characterisation of cell wall polymers from the heavily lignified tissues of olive (Olea europaea) seed hull

Cell wall material (CWM) was prepared from olive seed hull, which is heavily lignified and very tough. The material was cryomilled and delignified with chlorite/acetic acid for 9 h to give the holocellulose. Polymers were solubilised from the holocellulose by sequential extraction with cyclohexane-trans-1,2-diamine-NNN'N'-tetra-acetate (CDTA, Na salt), DMSO, 0.5, 1 and 4 KOH and 4 KOH + borate to leave the -cellulose residue. The suspension of -cellulose on neutralisation released a small amount of pectic material virtually free of xylan to give '-cellulose. The polymers from the various extracts were fractionated by graded precipitation with ethanol prior to anion-exchange chromatography, and selected fractions were subjected to methylation analysis. During delignification, glucuronoxylans with relatively low degrees of polymerisation (DP) and xylan-pectic polysaccharide complexes linked to degraded lignin were solubilised. A proportion of the xylan-pectic polysaccharide complexes were solubilised by 0.5 KOH. The major hemicellulosic polysaccharides of the olive seed hulls are glucuronoxylans, which occur as highly branched short chains, with DP of 30–60; or slightly branched chains with DP of 90–110. Partial acid hydrolysis of the major acidic xylan, gel-filtration chromatography and methylation analysis allowed us to propose a tentative structure for the major glucuronoxylan in which one residue of GlcpA occurs in each 14 continuously linked Xylp residues in a regular structure.     

Black piedra among the Zoró Indians from Amazônia (Brazil)

A total of 130 Zoró Indians from the Brazilian Amazon were observed as part of an epidemiological survey. Black piedra was found in 74 (56.9%) individuals. Infection rates between the sexes were not significantly different. The age group least infected comprised the children, 0–10 years of age. The authors comment on the epidemiology of the infection among the Zoró.     

Intravenous phage display identifies peptide sequences that target the burn-injured intestine

The injured intestine is responsible for significant morbidity and mortality after severe trauma and burn; however, targeting the intestine with therapeutics aimed at decreasing injury has proven difficult. We hypothesized that we could use intravenous phage display technology to identify peptide sequences that target the injured intestinal mucosa in a murine model, and then confirm the cross-reactivity of this peptide sequence with ex vivo human gut. Four hours following 30% TBSA burn we performed an in vivo, intravenous systemic administration of phage library containing 10¹² phage in balb/c mice to biopan for gut-targeting peptides. In vivo assessment of the candidate peptide sequences identified after 4 rounds of internalization was performed by injecting 1 × 10¹² copies of each selected phage clone into sham or burned animals. Internalization into the gut was assessed using quantitative polymerase chain reaction. We then incubated this gut-targeting peptide sequence with human intestine and visualized fluorescence using confocal microscopy. We identified 3 gut-targeting peptide sequences which caused collapse of the phage library (4–1: SGHQLLLNKMP, 4–5: ILANDLTAPGPR, 4–11: SFKPSGLPAQSL). Sequence 4–5 was internalized into the intestinal mucosa of burned animals 9.3-fold higher than sham animals injected with the same sequence (2.9 × 10⁵vs. 3.1 × 10⁴ particles per mg tissue). Sequences 4–1 and 4–11 were both internalized into the gut, but did not demonstrate specificity for the injured mucosa. Phage sequence 4–11 demonstrated cross-reactivity with human intestine. In the future, this gut-targeting peptide sequence could serve as a platform for the delivery of biotherapeutics.     

Gold complexes with benzimidazole derivatives: synthesis,characterization and biological studies

Synthesis, characterization, DFT studies and biological assays of new gold(I) and gold(III) complexes of benzimidazole are reported. Molecular and structural characterizations of the compounds were based on elemental (C, H and N) and thermal (TG–DTA) analyses, and FT-IR and UV–Visible spectroscopic measurements. The structures of complexes were proposed based DFT calculations. The benzimidazole compounds (Lig1 and Lig2) and the gold complexes were tested against three Leishmania species related to cutaneous manifestations of leishmaniasis. The free benzimidazole compounds showed no leishmanicidal activity. On the other hand, the gold(I and III) complexes have shown to possess significant activity against Leishmania in both stages of parasite, and the gold(III) complex with Lig2 exhibited expressive leishmanicidal activity with IC₅₀ values below 5.7 μM. Also, the gold complexes showed high leishmania selectivity. The gold(I) complex with Lig1, for example, is almost 50 times more toxic for the parasite than for macrophages. Besides the leishmanicidal activity, all complexes exhibited toxic effect against SK-Mel 103 and Balb/c 3T3, cancer cells.     

Biostratigraphy and paleoceanographical significance of the Neogene planktonic foraminifera from the Pelotas Basin, southernmost Brazil

Analysis of the planktonic foraminifers recovered from 1-SCS-2 drill-hole, Florianópolis platform (Pelotas Basin, southern Brazilian Atlantic Margin), allowed recognition of the Catapsydrax dissimilis, Catapsydrax stainforthi, Globorotalia fohsi robusta, Globorotalia mayeri, Globorotalia margaritae evoluta and Globigerinoides trilobus fistulosus Miocene and Pliocene zones and four important hiatuses. Correlation with well 1-SCS-3B and other zonal schemes, as well as recognition of early diagenesis instead of a reworking process, allowed confident age assignment. The Miocene foraminifers constitute a tropical/sub-tropical assemblage, whereas in the Pliocene, the presence of scarce species associated to subantarctic water masses suggests that the Malvinas Current reaches the area but it was not a controlling factor in the paleoenvironmental conditions.     

Effects of fungus inoculation and salt stress on physiology and biochemistry of in vitro grapevines: Emphasis on sugar composition changes by FT-IR analyses

The impacts of salt stress and inoculation in in vitro grapevine (Vitis vinifera L.) growth, nutrient accumulation, osmoregulation, photosynthesis and membrane integrity were evaluated. One month exposure to 100 mM NaCl as well as to inoculation with Phaeomoniella chlamydospora reduced relative growth rate (RGR) and induced senescence in grapevine plants, shown by: (1) decrease of Ψ_π without osmoregulation, (2) decrease of chlorophyll content and fluorescence, (3) loss of membrane integrity and (4) nutritional disorders. To assess putative changes in structural and/or non-structural carbohydrates induced by these two stress conditions, alcohol insoluble residues from the roots, stems and leaves were also characterised by FT-IR and GC with respect to the sugar composition. The referred organs were distinguished based on: (1) higher proportion of uronic acid residues in leaves which diagnose the presence of pectic polysaccharides (wavenumbers 1100, 1150 and 1018 cm^?1 in FT-IR spectra), (2) higher proportion of xylose and glucose on stems and FT-IR spectra diagnostic of xylose-rich polysaccharides (1041 cm^?1) and cellulose (1060 cm^?1), (3) higher proportion of glucose residues, xylose and arabinose on roots and a FT-IR spectra characteristic of xylose-rich polysaccharides (1041 cm^?1). The main alterations induced by salt stress and inoculation were more visible in leaves, where the content of uronic acid decreased showing that changes in cell wall composition occurred, mostly at the pectic fraction. Besides, an accumulation of insoluble glucose was found, and FT-IR spectra showed that this glucose-based material was starch (maximum absorption at 998 cm^?1), accumulated as a non-specific response to salt stress and P. chlamydospora inoculation.     

Partitioning of glycomacropeptide in aqueous two-phase systems

The partition behavior of glycomacropeptide (GMP) was determined in polyethylene glycol (PEG) and sodium citrate aqueous two-phase systems (ATPS). It was found that the partitioning of GMP depends on PEG molar mass, tie line length, pH, NaCl concentration and temperature. The obtained data indicates that GMP is preferentially partitioned into the PEG phase without addition of NaCl at pH 8.0. Larger tie line lengths and higher temperatures favor GMP partition to the PEG phase. Furthermore, it was verified that PEG molar mass and concentration have a slight effect on GMP partition. The increase in the molar mass of PEG induces a reduction of the protein solubility in the top PEG rich phase, being shown that the use of PEG1500 is beneficial for the extraction of GMP. A protein recovery higher than 85% was obtained in the top phase of these systems, clearly demonstrating its suitability as a starting point for the separation of GMP.     

Bioaccumulation of Amylose-Like Glycans by Helicobacter pylori

Background:  Helicobacter pylori cell surface is composed of lipopolysaccharides (LPSs) yielding structures homologous to mammalian Lewis O -chains blood group antigens. These structures are key mediators in the definition of host-microbial interactions and known to change their expression pattern in response to environmental pressure.
Aims:  The present work is focused on the identification of new H. pylori cell-surface glycosides. Special attention is further devoted to provide insights on the impact of in vitro subcultivation on H. pylori cell-surface phenotypes.
Methods:  Cell-surface glycans from H. pylori NCTC 11637 and two clinical isolates were recovered from the aqueous phase resulting from phenol:water extraction of intact bacteria. They were evaluated in relation to their sugars and glycosidic-linkages composition by CG-MS, size-exclusion chromatography, NMR, and Mass Spectrometry. H. pylori glycan profile was also monitored during subcultivation in vitro in agar and F12 liquid medium.
Results:  All three studied strains produce LPS expressing Lewis epitopes and express bioaccumulate amylose-like glycans. Bioaccumulation of amylose was found to be enhanced with the subcultivation of the bacterium on agar medium and accompanied by a decrease in the expression of LPS O -chains. In contrast, during exponential growth in F12 liquid medium, an opposite behavior is observed, that is, there is an increase in the overall amount of LPS and decrease in amylose content.
Conclusions:  This work shows that under specific environmental conditions, H. pylori expresses a phase-variable cell-surface α-(1→4)-glucose moiety.