Binding of hydrolases from wheat aleurone to concanavalin A- and wheat-germ agglutinin-sepharose

We have studied the ability of hydrolases (acid phosphatase and glycosidases) from the aleurone layers of resting wheat grains to interact with Con A- and WGA-Sepharose as a way to examine their glycoprotein nature. Aliquots (6–85% depending on the enzyme) of all the enzymes interacted with Con A-Sepharose. The major part of α-mannosidase activity (85%) was present in this form. Aliquots (2–20% depending on the enzyme) of the following four enzymes, β-galactosidase, α-mannosidase, β-N-acetylglucosaminidase and acid phosphatase, interacted with WGA-Sepharose. All the enzymes were found in forms which were unable to interact with either lectin. No forms of hydrolases interacting with both lectins were found in the crude extract. The specific activities of most of the enzymes recovered from the lectin-Sepharose gels were greater than those measured in the crude extract. In particular, the highest specific activities were found for β-N-acetylglucosaminidase and β-galactosidase recovered from WGA-Sepharose. Different lectin-binding forms of hydrolases were compared with respect to pH optimum and stability under various conditions (heat and guanidine hydrochloride treatments). The lectin-binding pattern of the hydrolases released in the incubation medium by the aleurone layers was similar to that reported above for the enzymes extracted from these tissues, suggesting that none of the hydrolase forms found in the aleurone layers is selectively released during incubation of these tissues.     

Tissue- and species-specific expression patterns of class I,III, and IV Adh and Aldh1 mRNAs in rodent embryos

Alcohol and aldehyde dehydrogenases (ADHs and ALDHs) may be of interest in the pathology of Parkinson's disease (PD) because of their role in protection against toxins and in retinoid metabolism, which is required for growth and development of the mesencephalic dopamine system. In the present study, the spatial and temporal expression patterns of Adh1, Adh3, Adh4, and Aldh1 mRNAs in embryonic C57BL/6 mice (E9.5-E19.5) and Sprague-Dawley rats (E12.5-P0) have been investigated by using radioactive oligonucleotide in situ hybridization. High expression of Aldh1 mRNA was found in the developing mesencephalic dopamine neurons of both mice and rats. Expression of Adh1 and Adh4 mRNAs was observed in adrenal cortex and olfactory epithelium in mice. Additionally, Adh1 was expressed in epidermis, liver, conjunctival, and intestinal epithelium. In rat embryos, expression was less extensive, with Adh1 mRNA being found in liver and intestines. Adh3 expression was ubiquitous in both mouse and rat embryos, suggesting a housekeeping function of the gene. Consistent with previous studies in adult rats and mice, our data suggest that Adh3 is the only ADH class present in rodent brain. Adh and Aldh gene activity in mouse and rat embryos indicate the possible involvement of the respective enzymes in retinoid metabolism and participation in defense against toxic insults, including those that may be involved in the pathogenesis of PD. This work was supported by grants from the Swedish Research Council, the Swedish Parkinson Foundation, the Swedish Brain Foundation, Karolinska Institutet funds, AstraZeneca, and the US Public Health Service.     

Replacement of K-Ras with H-Ras supports normal embryonic development despite inducing cardiovascular pathology in adult mice

Ras proteins are highly related GTPases that have key roles in regulating growth, differentiation and tumorigenesis. Gene-targeting experiments have shown that, out of the three mammalian ras genes, only K-ras is essential for normal mouse embryogenesis, and that mice deprived of H-ras and/or N-ras show no major phenotype. We generated mice (HrasKI) in which the K-ras gene had been modified to encode H-Ras protein. HrasKI mice produce undetectable amounts of K-Ras but-in contrast to mice homozygous for a null K-ras allele-they are born at the expected mendelian frequency, indicating that H-Ras can be substituted for K-Ras in embryonic development. However, adult HrasKI mice show dilated cardiomyopathy associated with arterial hypertension. Our results show that K-Ras can be replaced by H-Ras in its essential function in embryogenesis, and indicate that K-Ras has a unique role in cardiovascular homeostasis.     

Multilevel examination of minor salivary gland biopsy for Sjogren's syndrome significantly improves diagnostic performance of AECG classification criteria

The recently observed low reproducibility of focus score (FS) assessment at different section depths in a series of single minor salivary gland biopsies highlighted the need for a standardized protocol of extensive histopathological examination of such biopsies in Sjogren's syndrome. For this purpose, a cumulative focus score (cFS) was evaluated on three slides cut at 200-mum intervals from each of a series of 120 salivary biopsies. The cFS was substituted for the baseline FS in the American-European Consensus Group (AECG) criteria set for Sjogren's syndrome classification, and then test specificity and sensitivity were assessed against clinical patient re-evaluation. Test performances of the AECG classification with the original FS and the score obtained after multilevel examination were statistically compared using receiver operating characteristic (ROC) curve analysis. The diagnostic performance of AECG classification significantly improved when the cFS was entered in the AECG classification; the improvement was mostly due to increased specificity in biopsies with a baseline FS >or= 1 but <2. The assessment of a cFS obtained at three different section levels on minor salivary gland biopsies can be useful especially in biopsies with baseline FSs between 1 and 2.     

Crinipellis brasiliensis, a new species based on morphological and molecular data

Crinipellis perniciosa infects a diversity of hosts causing severe damage to T. cacao production in many Brazilian growing regions. We compared isolates of Crinipellis from different geographic origins and hosts in Brazil by structural analysis using light (LM) and scanning electronic microscopy (SEM), as well as RFLP and sequence data based on the nuclear rDNA ITS region. Statistical analyses of morphometric data of basidia and basidiospores revealed a distinct group of isolates of Crinipellis obtained from Heteropterys acutifolia when compared to representatives from Theobroma cacao, Solanum lycocarpum and Heteropterys nervosa. A similar distinction also was observed based on sequence data of the ITS region such that combined results allowed for the segregation of a new species within the genus Crinipellis.     

Mechanisms of soluble beta-amyloid impairment of endothelial function

Alzheimer's disease (AD) has been recently associated with vascular risk factors. beta-amyloid peptides (AbetaP), the main component of senile plaques typical of AD, circulate in soluble globular form in bloodstream. Interestingly, AbetaP is able to induce endothelial dysfunction, and this effect may represent the link between vascular and neuronal pathophysiological factors involved in AD. We aimed to clarify the molecular mechanisms underlying globular AbetaP-induced vascular toxicity. Using several methodological approaches, we have observed that in vascular tissues globular AbetaP is unable to induce oxidative stress, one of the mechanisms hypothesized involved in beta-amyloid toxicity. More important, we have demonstrated that globular AbetaP is able to localize on vascular endothelium, where it inhibits eNOS enzymatic activity. In particular, AbetaP enhances eNOS phosphorylation on threonine 495 and serine 116 and reduces acetylcholine-induced phosphorylation on serine 1177. Such an effect depends on a PKC signaling pathway, as suggested by its phosphorylation on serine 660. In fact, selective inhibition of the calcium-dependent group of PKC is able to rescue beta-amyloid-induced alteration of eNOS phosphorylation, NO production, and endothelial vasorelaxation. The activation of these Ca(2+)-dependent pathways is probably due to the ability of AbetaP to evoke Ca(2+) leakage from inositol 1,4,5-triphosphate receptors on endoplasmic reticulum. Our data demonstrate that globular AbetaP-induced endothelial NO dysfunction can be attributed to an alteration of intracellular Ca(2+) homeostasis, which could lead to the activation of calcium-dependent group of PKC with a consequent change of the eNOS phosphorylation pattern. These mechanisms could contribute to shed further light on the toxic effect of beta-amyloid in vascular tissues.     

Expression of glutathione S-transferase and peptide methionine sulphoxide reductase in Ochrobactrum anthropi is correlated to the production of reactive oxygen species caused by aromatic substrates

Peptide methionine sulphoxide reductase (MsrA) and glutathione S-transferases (GSTs) are considered as detoxification enzymes. In the xenobiotics-degrading bacterium Ochrobactrum anthropi the two enzymes are co-induced by toxic concentrations of aromatic substrates such as phenol and 4-chlorophenol. In aerobic organisms, degradation of aromatic substrates by mono- and dioxygenases leads to a generation of oxidative stress that causes the occurrence of reactive oxygen species (ROS). A capillary electrophoretic method, using the intracellular conversion of dihydrorhodamine-123 into rhodamine-123, was developed to measure the content of ROS in the bacteria. The presence of toxic concentrations of the aromatic substrate 4-chlorophenol, an inducer of GST and MsrA, leads to a significant increase in the production of ROS. These results strongly suggest that GST and MsrA enzymes are part of the bacterial defence mechanism against particular oxidative stress conditions. As oxidative stress is known to be present predominantly close to the cytoplasmic membrane, we investigated the subcellular distribution of both MsrA and GST enzymes in this bacterium grown in the presence of 4-chlorophenol. By Western blotting, MsrA and GST was assayed in the cytoplasm as well as in the periplasm. Moreover, immunolocalisation by colloidal gold immunoelectron microscopy identified the two proteins associated with the cell envelope.     

Evidence for δ-Opioid Binding and GTP-Regulatory Proteins in 5-Day-Old Rat Brain Membranes

The availability of the bispenicillamine enkephalin [3H] [D-Pen2,D-Pen5]enkephalin ([3H]DPDPE) a highly selective ligand for delta-opioid receptors, has made possible a more definitive examination of the ontogeny of this receptor subtype. In this report, the binding characteristics of [3H]DPDPE in 5-day-old neonatal (P-5) and adult rat brain are compared. Analysis of saturation curves as well as homologous displacement data revealed no significant difference in the binding affinity of [3H]DPDPE between P-5 animals and adults. Conversely, the binding capacity increased fivefold during this period. The delta-specificity of the sites was further proven by competition experiments with mu- and delta-selective ligands. Mn2+ (0.5 mM) elevated [3H]DPDPE specific binding by lowering the Kd, whereas 50 microM 5'-guanylylimidodiphosphate inhibited it by decreasing the total number of high-affinity binding sites in both P-5 animals and adults. Pertussis toxin-catalyzed ADP ribosylation experiments revealed the presence of 40-kDa proteins, with a molecular mass corresponding to G protein subunits alpha i/alpha o, as early as 1 h after birth. There was a low, but detectable, basal low-Km GTPase activity in P-5 animals, which increased fivefold during postnatal development. The present report establishes the existence of high-affinity [3H]DPDPE binding as well as GTP-regulatory proteins 5 days after birth. Yet, heterologous competition studies and ionic effects suggest that neonatal binding sites differ from adult receptors. Whether the neonatal sites are newly synthesized, incompletely processed sites or a developmentally programmed isoform remains to be determined.     

RETRACTED ARTICLE: Further in vitro biological activity evaluation of amino-, thio- and ester-derivatives of avarol

The acetylcholinesterase inhibitory and/or antitumour activities of amino-, thio- and ester-derivatives of avarol selected were evaluated for the first time at in vitro conditions. Avarol-3′,4′-dithioglycol (1) and avarol-4′-(3)mercaptopropionic acid (3) were shown to be the best inhibitors of the enzyme tested (0.50?µg and IC50 0.05?mM and 0.50?µg and IC50 0.12?mM, respectively), while 4′-tryptamine-avarone (9) and avarol-3′-(3)mercaptopropionic acid (2) exhibited the highest cytotoxicity against the human breast T-47D cancer cell line (IC50 0.66?µg/mL and 1.25?µg/mL, respectively). According to experimental data obtained, the sesquiterpenoid hydroquinone structure of bioactive avarol derivatives may inspire development of new pharmacologically useful substances to be used in the treatment of Alzheimer's disease and/or human breast tumour.