Aldehyde oxidase (AO) in the root nodules of Lupinus albus and Medicago truncatula: identification of AO in meristematic and infection zones

Phytohormones are involved in the organogenesis of legume root nodules. The source of the auxin indole-3-acetic acid (IAA) in nodules has not been clearly determined. We studied the enzyme aldehyde oxidase (AO; EC 1.2.3.1), that catalyzes the last step of IAA biosynthesis in plants, in the nodules of Lupinus albus and Medicago truncatula. Primordia and young lupin nodules and mature M. truncatula nodules showed AO activity bands after native polyacrylamide gel electrophoresis. Gel activity analyses using indole-3-aldehyde as substrate indicated that the nodules of white lupin and M. truncatula have the capability to synthesize IAA via the indole-3-pyruvic acid pathway. Immunolocalization and in situ hybridization experiments revealed that AO is preferentially expressed in the meristematic and the invasion zones in Medicago nodules and in the lateral meristematic zone of Lupinus nodules. High IAA immunolabeling was also detected in the meristematic and invasion zones. Low expression levels and no AO activity were detected in lupin Fix- nodules that displayed restricted growth and early senescence. We propose that local synthesis of IAA in the root nodule meristem and modulation of AO expression and activity are involved in regulation of nodule development.     

Pathogen adaptation to seasonal forcing and climate change

Many diverse infectious diseases exhibit seasonal dynamics. Seasonality in disease incidence has been attributed to seasonal changes in pathogen transmission rates, resulting from fluctuations in extrinsic climate factors. Multi-strain infectious diseases with strain-specific seasonal signatures, such as cholera, indicate that a range of seasonal patterns in transmission rates is possible in identical environments. We therefore consider pathogens capable of evolving their 'seasonal phenotype', a trait that determines the sensitivity of their transmission rates to environmental variability. We introduce a theoretical framework, based on adaptive dynamics, for predicting the evolution of disease dynamics in seasonal environments. Changes in the seasonality of environmental factors are one important avenue for the effects of climate change on disease. This model also provides a framework for examining these effects on pathogen evolution and associated disease dynamics. An application of this approach gives an explanation for the recent cholera strain replacement in Bangladesh, based on changes in monsoon rainfall patterns.     

Angiotensin II induces focal adhesion kinase/paxillin phosphorylation and cell migration in human umbilical vein endothelial cells

In the present study, we demonstrated that Ang II provokes a transitory enhancement of focal adhesion kinase (FAK) and paxillin phosphorylation in human umbilical endothelial cells (HUVEC). Moreover, Ang II induces a time- and dose-dependent augmentation in cell migration, but does not affect HUVEC proliferation. The effect of Ang II on FAK and paxillin phosphorylation was markedly attenuated in cells pretreated with wortmannin and LY294002, indicating that phosphoinositide 3-kinase (PI3K) plays an important role in regulating FAK activation. Similar results were observed when HUVEC were pretreated with genistein, a non-selective tyrosine kinases inhibitor, or with the specific inhibitor PP2 for Src family kinases, demonstrating the involvement of protein tyrosine kinases, and particularly Src family of tyrosine kinases, in the downstream signalling pathway of Ang II receptors. Furthermore, FAK and paxillin phosphorylation was markedly blocked after treatment of HUVEC with AG1478, a selective inhibitor of epidermal growth factor receptor (EGFR) phosphorylation. Pretreatment of cells with inhibitors of PI3K, Src family tyrosine kinases, and EGFR also decreased HUVEC migration. In conclusion, these results suggest that Ang II mediates an increase in FAK and paxillin phosphorylation and induces HUVEC migration through signal transduction pathways dependent on PI3K and Src tyrosine kinase activation and EGFR transactivation.     

Two L1-peptides are excellent tools for serological detection of HPV-associated cervical carcinoma lesions

A persistent high risk human papillomavirus (HR-HPV) infection causes cervical intraepithelial lesions and cervical carcinoma. There is evidence that detecting anti-L1 antibodies could be successfully used for discriminating between cervical lesion patients and women having normal cytology. It was found that peptides 18283 (55PNNNKILVPKVSGLQYRVFR74) and 18294 (284LYIKGSGSTANLASSNYFPT300) from the L1-surface exposed regions were specifically recognised by antibodies from the cervical lesion patient sera. These peptides were tested against 165 womens' normal cytology sera and 148 cervical lesion or cervical cancer patients' sera. Less than 3.6% of women's normal cytology sera recognised peptides 18283 or 18294; on the contrary, 91% to 96% of the cervical lesion (CIN I to CIN III) or cervical cancer patient sera recognised peptides 18283 and 18294. These data show that anti-peptide 18283 and 18294 antibodies in the patients' sera are strongly associated with the presence of HR-HPV associated cervical lesions, showing 92-97% sensitivity and 89-95% specificity in recognising precancerous and cervical cancer patients. These two peptides could be excellent tools for use in large-scale serological screening of women populations at risk of developing cervical carcinoma.     

Defective CD3zeta chain expression in Herpesvirus saimiri (HVS)-derived T-cell lines in gastric adenocarcinoma

Low expression of the CD3zeta chain has been reported in patients with cancer and it has been suggested that tumor-derived factors are involved in its downregulation. The expression of CD3zeta chain was measured in T-cell lines from patients with gastric adenocarcinoma and healthy volunteers and grown in vitro for several months and, hence, in the absence of any tumor-derived factors. T-cell lines of mucosal origin were obtained by Herpesvirus saimiri transformation from gastric cancer patients. The expression of CD3zeta and CD3epsilon was measured by flow cytometry and Western-blot analysis. Calcium mobilization and apoptosis rate were also measured. The levels of CD3zeta, but not CD3epsilon, chain on the cell surface were significantly reduced in T-cell lines derived from patients with gastric cancer when cultured in the absence of IL-2. Western-blot analysis of total cell extracts or lipid raft fractions confirmed this finding. Calcium mobilization, a measure of signal transduction, was reduced in T cell lines from patients with gastric cancer. We conclude that T cells from patients with cancer express lower levels of CD3zeta. This downregulation is not caused by a direct effect of tumor-derived factors but, rather, it appears to be inherent to the patient cells. The low CD3zeta expression would render T lymphocytes unable to control the growth of tumor cells.     

Characterization of acyl-phosphatidylinositol from the opportunistic pathogen Corynebacterium amycolatum

The aim of the present study was to characterize a new lipid detected in the opportunistic pathogen Corynebacterium amycolatum. It was identified as acyl-phosphatidylinositol (acyl-PI), and revealed as a mixture of homologues compounds by electrospray ionization mass spectrometry, with pseudomolecular ions, (M-H)-, observed at 1099 (the major one) 1113, and 1127. Acyl-PI exclusively contained octadecenoyl on the inositol moiety (as 3-O-acyl), an unsaturated fatty acyl (mostly octadecenoyl) at sn-1 position of the glycerol and a saturated fatty acyl (mainly hexadecanoyl) at the sn-2 position. Acyl-PI constitutes a new natural substance and seems to be unique among the phospholipids of C. amycolatum. Other more complex molecules, previously undetected, and assigned in this work to several acyl forms of phosphatidylinositol trimannosides, lacked octadecenoyl in their polar heads. The present study reveals the existence of acyl-PI in C. amycolatum as rather unexpected finding and, additionally, gives evidence for the ability of this species to synthesize a great variety of inositol-containing phospholipids.     

Evidence that 17alpha-estradiol is biologically active in the uterine tissue: Antiuterotonic and antiuterotrophic action

Background  

17alpha-Estradiol has been considered as the hormonally inactive isomer of 17beta-estradiol. Recently, nongenomic (smooth muscle relaxation) and genomic (light estrogenic activity) effects of 17alpha-estradiol have been reported, but no reports have yet determined its possible antiestrogenic activity. Therefore, this study investigated: the nongenomic action of 17alpha-estradiol on uterine contractile activity and its potential agonist-antagonist activity on uterine growth.     

Production and characterization of a noncytotoxic deletion variant of the Aspergillus fumigatus allergen Aspf1 displaying reduced IgE binding

Aspergillus fumigatus is responsible for many allergic respiratory diseases, the most notable of which - due to its severity - is allergic bronchopulmonary aspergillosis. Aspf1 is a major allergen of this fungus: this 149-amino acid protein belongs to the ribotoxin family, whose best characterized member is alpha-sarcin (EC 3.1.27.10). The proteins of this group are cytotoxic ribonucleases that degrade a unique bond in ribosomal RNA impairing protein biosynthesis. Aspf1 and its deletion mutant Aspf1Delta(7-22) have been produced as recombinant proteins; the deleted region corresponds to an exposed beta-hairpin. The conformation of these two proteins has been studied by CD and fluorescence spectroscopy. Their enzymatic activity and cytotoxicity against human rhabdomyosarcoma cells was also measured and their allergenic properties have been studied by using 58 individual sera of patients sensitized to Aspergillus. Aspf1Delta(7-22) lacks cytotoxicity and shows a remarkably reduced IgE reactivity. From these studies it can be concluded that the deleted beta-hairpin is involved in ribosome recognition and is a significant allergenic region.     

The seventh transmembrane domains of the delta and kappa opioid receptors have different accessibility patterns and interhelical interactions

We applied the substituted cysteine accessibility method (SCAM) to map the residues of the transmembrane helices (TMs) 7 of delta and kappa opioid receptors (deltaOR and kappaOR) that are on the water-accessible surface of the binding-site crevices. A total of 25 consecutive residues (except C7.38) in the TMs 7 were mutated to Cys, one at a time, and each mutant was expressed in HEK 293 cells. Most mutants displayed similar binding affinity for [(3)H]diprenorphine, an antagonist, as the wild types. Pretreatment with (2-aminoethyl)methanethiosulfonate (MTSEA) inhibited [(3)H]diprenorphine binding to eight deltaOR and eight kappaOR mutants. All mutants except deltaOR L7.52(317)C were protected by naloxone from the MTSEA effect, indicating that the side chains of V7.31(296), A7.34(299), I7.39(304), L7.41(306), G7.42(307), P7.50(315), and Y7.53(318) of deltaOR and S7.34(311), F7.37(314), I7.39(316), A7.40(317), L7.41(318), G7.42(319), Y7.43(320), and N7.49(326) of kappaOR are on the water-accessible surface of the binding pockets. Combining the SCAM data with rhodopsin-based molecular models of the receptors led to the following conclusions. (i) The residues of the extracellular portion of TM7 predicted to face TM1 are sensitive to MTSEA in kappaOR but are not in deltaOR. Thus, TM1 may be closer to TM7 in deltaOR than in kappaOR. (ii) MTSEA-sensitive mutants start at position 7.31(296) in deltaOR and at 7.34(311) in kappaOR, suggesting that TM7 in deltaOR may have an additional helical turn (from 7.30 to 7.33). (iii) There is a conserved hydrogen-bond network linking D2.50 of the NLxxxD motif in TM2 with W6.48 of the CWxP motif in TM6. (iv) The NPxxY motif in TM7 interacts with TM2, TM6, and helix 8 to maintain receptors in inactive states. To the best of our knowledge, this represents the first such comparison of the structures of two highly homologous GPCRs.     

Quantification of yessotoxin using the fluorescence polarization technique and study of the adequate extraction procedure

Yessotoxin (YTX) is a polycyclic ether toxin produced by phytoplanktonic microalgae from the group of dinoflagellates. It has been shown that YTX increases the 3',5'-cyclic nucleotide phosphodiesterases (PDEs) activity and that there is a binding between these proteins and the toxin. Fluorescence polarization (FP) is a spectroscopic technique that can be used to study the interactions between molecules. It is based on exciting a fluorescent molecule with plane-polarized light and measuring the polarization degree of the emitted light. In this study, the FP is applied to the study of the interaction between YTX and phosphodiesterases I and II (PDE I and II). The phosphodiesterases are labeled with a reactive succinimidyl esther of carboxyfluorescein, and the FP of the protein-dye conjugate is measured when the YTX concentration in the medium increases. The results show that in both cases the fluorescence polarization of the conjugates decreases when they bind to YTX. For the PDE I, it is possible to draw a Gaussian curve or a straight line that relates the two variables (FP and YTX concentration). The concentration of this toxin in a spiked mussel extract (which contains the conjugate) can be quantified measuring its FP and using the equations of those lines. Different extraction methods are tried in this study, and those that can be used to obtain an appropriate mussel extract to be quantified with this technique are determined.