Structural homology of storage proteins coded by the Hor-1 locus of barley (Hordeum vulgare L.)

Three C hordein fractions were prepared by ion-exchange chromatography of a total hordein preparation on carboxymethyl cellulose at pH 4.6 Polyacrylamide gel electrophoresis at pH 3.2 and sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) at pH 8.9 showed that each fraction contained a single major band. The apparent molecular weights of these were determined by SDS-PAGE as 58, 57, and 54,000. When compared by isoelectric focusing, however, the 58 and 57,000 components each separated into two major bands and the 54,000 component into four. Amino acid analysis showed that although the three fractions had similar compositions with high glutamate+glutamine (38–39%), proline (30–32%) and phenylalanine (8–9%) contents, some differences were present, notably in the relative content of lysine. The three fractions had identical amino acid sequences for the first ten residues at the N-terminal end. They also had identical sequences for the first five residues at the C-terminal end, with the exception that a mixture of two amino acids were released from position 4 of the 58,000 fraction only. Peptide mapping with three enzymes (trypsin, chymotrypsin and V8 protease) indicated that the 58 and 57,000 fractions were more closely related to each other than to the 54,000 fraction. It is suggested that the 57 and 58,000 fractions and the 54,000 fraction constitute two families of closely related polypeptides which are coded by genes derived from the duplication and divergence of a single ancestral gene.     

Autophagy inhibition radiosensitizes in vitro,yet reduces radioresponses in vivo due to deficient immunogenic signalling

Clinical oncology heavily relies on the use of radiotherapy, which often leads to merely transient responses that are followed by local or distant relapse. The molecular mechanisms explaining radioresistance are largely elusive. Here, we identified a dual role of autophagy in the response of cancer cells to ionizing radiation. On one hand, we observed that the depletion of essential autophagy-relevant gene products, such as ATG5 and Beclin 1, increased the sensitivity of human or mouse cancer cell lines to irradiation, both in vitro (where autophagy inhibition increased radiation-induced cell death and decreased clonogenic survival) and in vivo, after transplantation of the cell lines into immunodeficient mice (where autophagy inhibition potentiated the tumour growth-inhibitory effect of radiotherapy). On the other hand, when tumour proficient or deficient for autophagy were implanted in immunocompetent mice, it turned out that defective autophagy reduced the efficacy of radiotherapy. Indeed, radiotherapy elicited an anti-cancer immune response that was dependent on autophagy-induced ATP release from stressed or dying tumour cells and was characterized by dense lymphocyte infiltration of the tumour bed. Intratumoural injection of an ecto-ATPase inhibitor restored the immune infiltration of autophagy-deficient tumours post radiotherapy and improved the growth-inhibitory effect of ionizing irradiation. Altogether, our results reveal that beyond its cytoprotective function, autophagy confers immunogenic properties to tumours, hence amplifying the efficacy of radiotherapy in an immunocompetent context. This has far-reaching implications for the development of pharmacological radiosensitizers.     

DDC- and 3TC-bis(SATE) Monophosphate Prodrugs Overcome Cellular Resistance Mechanisms to HIV-1 Associated with Cytidine Kinase Deficiency

Abstract

A 2,3′-dideoxycytidine (ddC)-resistant T-lymphoid cell line (MOLT-4/8^rddC²⁵⁰), in which deoxycytidine kinase (dCK) gene-expression was decreased when compared with parental cells, has been selected. Cytotoxic and antiretroviral activity of ddC and 3TC was significantly lower in MOLT-4/8^rddC²⁵⁰ than in parental MOLT-4/8 cells. ddC- and 3TC-bis(SATE)phosphotriesters completely overcame cellular resistance mechanisms and showed comparable both cytotoxic and antiretroviral activity in parental and ddC-resistant cells.     

Restricted access materials and large particle supports for on-line sample preparation: an attractive approach for biological fluids analysis

An analytical process generally involves four main steps: (1) sample preparation; (2) analytical separation; (3) detection; and (4) data handling. In the bioanalytical field, sample preparation is often considered as the time-limiting step. Indeed, the extraction techniques commonly used for biological matrices such as liquid-liquid extraction (LLE) and solid-phase extraction (SPE) are achieved in the off-line mode. In order to perform a high throughput analysis, efforts have been engaged in developing a faster sample purification process. Among different strategies, the introduction of special extraction sorbents, such as the restricted access media (RAM) and large particle supports (LPS), allowing the direct and repetitive injection of complex biological matrices, represents a very attractive approach. Integrated in a liquid chromatography (LC) system, these extraction supports lead to the automation, simplification and speeding up of the sample preparation process. In this paper, RAM and LPS are reviewed and particular attention is given to commercially available supports. Applications of these extraction supports, are presented in single column and column-switching configurations, for the direct analysis of compounds in various biological fluids.     

Localization of beta-D-glucosidase activity and glucovanillin in vanilla bean (Vanilla planifolia Andrews)

The morphology, anatomy and histology of mature green vanilla beans were examined by light and transmission electron microscopy. Beans have a triangular cross-section with a central cavity containing seeds. Each angle is lined with tubular cells, or papillae, while the cavity sides consist of placental laminae. The epicarp and endocarp are formed by one or two layers of very small cells, while the mesocarp contains large, highly vacuolarized cells, the cytoplasm being restricted to a thin layer along the cell walls. The radial distributions of glucovanillin and beta-glucosidase activity, measured on p-nitrophenyl-beta-glucopyranoside and glucovanillin, are superimposable and show how beta-glucosidase activity increases from the epicarp towards the placental zone, whereas glucovanillin is exclusively located in the placentae and papillae. Subcellular localization of beta-glucosidase activity was achieved by incubating sections of vanilla beans in a buffer containing 5-bromo-4-chloro-3-indolyl-beta-d-glucopyranoside as a substrate. Activity was observed in the cytoplasm (and/or the periplasm) of mesocarp and endocarp cells, with a more diffuse pattern observed in the papillae. A possible mechanism for the hydrolysis of glucovanillin and release of the aromatic aglycon vanillin involves the decompartmentation of cytoplasmic (and/or periplasmic) beta-glucosidase and vacuolar glucovanillin.     

Induction of apoptotic death in cells via Bad gene expression by infectious pancreatic necrosis virus infection

A Bcl-2 related family member, Bad, promotes cell death, and its function is regulated by phosphorylation. In this study, we show how the IPNV elicits the induction of Bad gene expression and promotes host apoptotic death. Anti-IPNV-E1S polyclonal and anti-VP3 monoclonal antibodies are used to neutralize the virus that blocks the prime death signal via the virus receptor. In the viability assay, each antibody could also enhance cell viability during IPNV infection. We tested tyrosine kinase inhibitors on IPNV-infected cells in order to assess their effect on blocking the death signal. With 100 microg/ml genistein treatment, Bad-like gene expression was blocked, either by rescuing the IPNV-infected CHSE-214 cells or by blocking internucleosomal DNA cleavage; but the tyrphostin group did not block Bad expression. For CHSE-214 cells, treatment with the protein synthesis-inhibitor, cycloheximide (1microg/ml), blocked new protein synthesis via activated tyrosine kinase during IPNV infection. We found that Bad protein expression could be blocked, and apoptotic death prevented. Together, these results demonstrate that the IPNV exerts up-regulation of a pro-apoptotic death gene (Bad), the expression of which serves to trigger apoptotic cell death. Our data also suggests that the IPNV induces apoptotic death via a viral receptor which triggers death effector Bad gene expression, possibly through a tyrosine kinase-dependent pathway.     

Positive regulation of apoptosis by HCA66, a new Apaf-1 interacting protein, and its putative role in the physiopathology of NF1 microdeletion syndrome patients

As a component of the apoptosome, a caspase-activating complex, Apaf-1 plays a central role in the mitochondrial caspase activation pathway of apoptosis. We report here the identification of a novel Apaf-1 interacting protein, hepatocellular carcinoma antigen 66 (HCA66) that is able to modulate selectively Apaf-1-dependent apoptosis through its direct association with the CED4 domain of Apaf-1. Expression of HCA66 was able to potentiate Apaf-1, but not receptor-mediated apoptosis, by increasing downstream caspase activity following cytochrome c release from the mitochondria. Conversely, cells depleted of HCA66 were severely impaired for apoptosome-dependent apoptosis. Interestingly, expression of the Apaf-1-interacting domain of HCA66 had the opposite effect of the full-length protein, interfering with the Apaf-1 apoptotic pathway. Using a cell-free system, we showed that reduction of HCA66 expression was associated with a diminished amount of caspase-9 in the apoptosome, resulting in a lower ability of the apoptosome to activate caspase-3. HCA66 maps to chromosome 17q11.2 and is among the genes heterozygously deleted in neurofibromatosis type 1 (NF1) microdeletion syndrome patients. These patients often have a distinct phenotype compared to other NF1 patients, including a more severe tumour burden. Our results suggest that reduced expression of HCA66, owing to haploinsufficiency of HCA66 gene, could render NF1 microdeleted patients-derived cells less susceptible to apoptosis.     

Spectroscopic description of an unusual protonated ferryl species in the catalase from <Emphasis Type="Italic">Proteus mirabilis</Emphasis> and density functional theory calculations on related models. Consequences for the ferryl protonation state in catalase,peroxidase and chloroperoxidase

The catalase from Proteus mirabilis peroxide-resistant bacteria is one of the most efficient heme-containing catalases. It forms a relatively stable compound II. We were able to prepare samples of compound II from P. mirabilis catalase enriched in ⁵⁷Fe and to study them by spectroscopic methods. Two different forms of compound II, namely, low-pH compound II (LpH II) and high-pH compound II (HpH II), have been characterized by Mössbauer, extended X-ray absorption fine structure (EXAFS) and UV-vis absorption spectroscopies. The proportions of the two forms are pH-dependent and the pH conversion between HpH II and LpH II is irreversible. Considering (1) the Mössbauer parameters evaluated for four related models by density functional theory methods, (2) the existence of two different Fe–O_ferryl bond lengths (1.80 and 1.66 Å) compatible with our EXAFS data and (3) the pH dependence of the α band to β band intensity ratio in the absorption spectra, we attribute the LpH II compound to a protonated ferryl Fe^IV–OH complex (Fe–O approximately 1.80 Å), whereas the HpH II compound corresponds to the classic ferryl Fe^IV=O complex (Fe=O approximately 1.66 Å). The large quadrupole splitting value of LpH II (measured 2.29 mm s^?1 vs. computed 2.15 mm s^?1) compared with that of HpH II (measured 1.47 mm s^?1 vs. computed 1.46 mm s^?1) reflects the protonation of the ferryl group. The relevancy and involvement of such (Fe^IV=O/Fe^IV–OH) species in the reactivity of catalase, peroxidase and chloroperoxidase are discussed.