Early ultrastructural injuries in the thyroid of the normal rat radioinduced by diagnostic and/or therapeutic amounts of iodine-131.

After irradiation, two principal mechanisms of cytolytic cell death can be involved: apoptosis and necrosis. By using morphological criteria, cells undergoing apoptosis can be distinguished from cells dying by necrosis. In nuclear medicine 131I is used to ablate thyroid remnants or to treat well differentiated thyroid carcinoma. The aim of study was to describe the progressive morphological thyroid changes induced by a diagnostic and/or therapeutic amounts of 131I in the rat using electron microscopy, in an attempt to determine which is the cell death pathway and to analyse "stunned" thyroid tissue to elucidate this effect. Tissular and ultrastructural examinations show that damages induced by 131I irradiation of the normal thyroid gland are heterogeneous. Thyroid cells die by necrosis after this metabolic irradiation, and no signs of apoptosis were observed by electron microscopy. In the other hand, stunning effect did not seem to impair the effectiveness of 131I treatment.     

Selective inhibition of dipeptidyl peptidase I,not caspases,prevents the partial processing of procaspase-3 in CD3-activated human CD8(+) T lymphocytes

Activation of primary human T cells by anti-CD3 and interleukin-2 resulted in partial processing of procaspase-3 in activated nonapoptotic (Delta Psi(m)high) CD8(+) T cells but not in CD4(+) T cells. Apical caspases-8 and -9 were not activated, and Bid was not processed to truncated Bid. Boc-D.fmk, a broad spectrum caspase inhibitor, did not prevent this process, whereas GF.dmk, a selective inhibitor of dipeptidyl peptidase I, was effective. Dipeptidyl peptidase I is required for the activation of granule-associated serine proteases. It is enriched in the cytolytic granules of cytotoxic lymphocytes, where it promotes the proteolytic activation of progranzymes A and B. Inhibition of granzyme B (GrB)-like serine proteases by Z-AAD.cmk prevented partial processing of procapase-3, whereas inhibition of GrA activity by D-FPR.cmk had no effect. Specific inhibitors of other lysosomal proteases such as cathepsins B, L, and D did not interfere in this event. Patients with Chediak-Higashi syndrome or with perforin deficiency also displayed partial processing of procaspase-3, excluding the involvement of granule exocytosis for the delivery of the serine protease in cause. The p20/p12 processing pattern of procaspase-3 in our model points to GrB, the sole serine protease with caspase activity. Small amounts of GrB were indeed exported from cytolytic granules to the cytosol of a significant fraction of GrB-positive cells.     

Mechanisms of arsenic hyperaccumulation in Pteris vittata. Uptake kinetics,interactions with phosphate,and arsenic speciation

The mechanisms of arsenic (As) hyperaccumulation in Pteris vittata, the first identified As hyperaccumulator, are unknown. We investigated the interactions of arsenate and phosphate on the uptake and distribution of As and phosphorus (P), and As speciation in P. vittata. In an 18-d hydroponic experiment with varying concentrations of arsenate and phosphate, P. vittata accumulated As in the fronds up to 27,000 mg As kg(-1) dry weight, and the frond As to root As concentration ratio varied between 1.3 and 6.7. Increasing phosphate supply decreased As uptake markedly, with the effect being greater on root As concentration than on shoot As concentration. Increasing arsenate supply decreased the P concentration in the roots, but not in the fronds. Presence of phosphate in the uptake solution decreased arsenate influx markedly, whereas P starvation for 8 d increased the maximum net influx by 2.5-fold. The rate of arsenite uptake was 10% of that for arsenate in the absence of phosphate. Neither P starvation nor the presence of phosphate affected arsenite uptake. Within 8 h, 50% to 78% of the As taken up was distributed to the fronds, with a higher translocation efficiency for arsenite than for arsenate. In fronds, 49% to 94% of the As was extracted with a phosphate buffer (pH 5.6). Speciation analysis using high-performance liquid chromatography-inductively coupled plasma mass spectroscopy showed that >85% of the extracted As was in the form of arsenite, and the remaining mostly as arsenate. We conclude that arsenate is taken up by P. vittata via the phosphate transporters, reduced to arsenite, and sequestered in the fronds primarily as As(III).     

Determination of residual dipolar couplings in homonuclear MOCCA-SIAM experiments

In solutions with partial molecular alignment, anisotropic magnetic interactions such as the chemical shift anisotropy, the electric quadrupole interaction, and the magnetic dipole-dipole interaction are no longer averaged out to zero in contrast to isotropic solutions. The resulting residual anisotropic magnetic interactions are increasingly used in biological NMR studies for the determination of 3D structures of proteins and other biomolecules. In the present paper we propose a new approach allowing the measurement of residual H^N-H dipolar couplings of non-isotope enriched proteins based on the application of the MOCCA-SIAM experiment. This experiment allows the measurement of homonuclear coupling constants with an accuracy of ca. ±0.2 Hz and is therefore particularly well suited to determine residual dipolar couplings at relatively low degrees of molecular orientation. The agreement between experimentally determined residual H^N-H couplings and calculated values is demonstrated for BPTI.     

Development of anti-TNF therapy for rheumatoid arthritis

The aetiology of systemic, autoimmune, chronic inflammatory diseases--such as rheumatoid arthritis--is not known, and their pathogenesis is complex and multifactorial. However, progress in the characterization of intercellular mediators--proteins that are now known as cytokines--has led to the realization that one cytokine, tumour-necrosis factor (TNF; previously known as TNF-alpha), has an important role in the pathogenesis of rheumatoid arthritis. This discovery heralded a new era of targeted and highly effective therapeutics for rheumatoid arthritis and, subsequently, other chronic inflammatory diseases.     

Mitochondrial permeability transition as a novel principle of hepatorenal toxicity in vivo

Atractyloside (Atr) binds to the adenine nucleotide translocator (ANT) and inhibits ANT-mediated ATP/ADP exchange on the inner mitochondrial membrane. In addition, Atr can trigger opening of a non-specific ion channel, within the ANT-containing permeability transition pore complex (PTPC), which is subject to redox regulation and inhibited by cyclosporin A (CsA). Here we show that the cytotoxic effects of Atr, both in vivo and in vitro, are determined by its capacity to induce PTPC opening and consequent mitochondrial membrane permeabilization (MMP). Thus, the Atr-induced MMP and death of cultured liver cells are both inhibited by CsA as well as by glutathione (GSH) and enhanced by GSH depletion. Similarly, the hepatorenal toxicity of Atr, assessed in vivo, was reduced by treating mice with CsA or a diet rich in sulfur amino acids, a regime which enhances mitochondrial GSH levels. Atr injection induced MMP in hepatocytes and proximal renal tubular cells, and MMP was reduced by either CsA or GSH. Acetaminophen (paracetamol)-induced acute poisoning was also attenuated by CsA and GSH, both in vitro and in vivo. Altogether these data indicate that PTPC-mediated MMP may determine the hepatorenal toxicity of xenobiotics in vivo.     

In vitro folding,functional characterization,and disulfide pattern of the extracellular domain of human GLP-1 receptor

The N-terminal, extracellular domain of the receptor for glucagon-like peptide 1 (GLP-1 receptor) was expressed at a high level in E. coli and isolated as inclusion bodies. Renaturation with concomitant disulfide bond formation was achieved from guanidinium-solubilized material. A soluble and active fraction of the protein was isolated by ion exchange chromatography and gel filtration. Complex formation with GLP-1 was shown by cross-linking experiments, surface plasmon resonance measurements, and isothermal titration calorimetry. The existence of disulfide bridges in the N-terminal receptor fragment was proven after digestion of the protein with pepsin. Further analysis revealed a disulfide-binding pattern with links between cysteines 46 and 71, 62 and 104, and between 85 and 126.     

A fluorescence microplate assay using yopro-1 to measure apoptosis: application to HL60 cells subjected to oxidative stress

A new one-step labeling procedure using the membrane permeant fluorescent probe yopro-1 in association with fluorescence microtitration for the rapid determination of apoptosis is reported. Programmed cell death was induced by the pro-apoptotic agents etoposide and staurosporine, and measured in nonadherent HL60 cells and adherent phorbol 12-myristate 13-acetate (PMA)-treated HL60 cells. Cell viability was controlled by trypan blue exclusion and calcein-AM staining. To confirm results of fluorescence microplate assay, apoptosis was measured by flow cytometry analysis using the same fluorescent probe, and results showed corresponding data between both procedures. Development of apoptosis was confirmed by the presence of PARP (poly(ADP-ribose) polymerase cleavage and nuclear DAPI (4,6-diamidino-2-phenylindole) staining, two well-known methods used to investigate apoptosis. The fluorescence microplate assay was also applied to measure apoptosis in cells exposed to an oxidative stress induced by tert-butylhydroperoxide (t-BHP), and results confirmed the potential of the fluorescence microplate assay in measuring events of apoptosis, especially in adherent, cultured, living cells.     

Munc13-4 is essential for cytolytic granules fusion and is mutated in a form of familial hemophagocytic lymphohistiocytosis (FHL3)

Secretion of cytolytic granules content at the immunological synapse is a highly regulated process essential for lymphocyte cytotoxicity. This process requires the rapid transfer of perforin containing lytic granules to the target cell interface, followed by their docking and fusion with the plasma membrane. Defective cytotoxicity characterizes a genetically heterogeneous condition named familial hemophagocytic lymphohistiocytosis (FHL), which can be associated with perforin deficiency. The locus of a perforin (+) FHL subtype (FHL3), observed in 10 patients, was mapped to 17q25. This region contains hMunc13-4, a member of the Munc13 family of proteins involved in vesicle priming function. HMunc13-4 mutations were shown to cause FHL3. HMunc13-4 deficiency results in defective cytolytic granule exocytosis, despite polarization of the secretory granules and docking with the plasma membrane. Expressed tagged hMunc13-4 localizes with cytotoxic granules at the immunological synapse. HMunc13-4 is therefore essential for the priming step of cytolytic granules secretion preceding vesicle membrane fusion.     

The Hansenula polymorpha (strain CBS4732) genome sequencing and analysis

The methylotrophic yeast Hansenula polymorpha is a recognised model system for investigation of peroxisomal function, special metabolic pathways like methanol metabolism, of nitrate assimilation or thermostability. Strain RB11, an odc1 derivative of the particular H. polymorpha isolate CBS4732 (synonymous to ATCC34438, NRRL-Y-5445, CCY38-22-2) has been developed as a platform for heterologous gene expression. The scientific and industrial significance of this organism is now being met by the characterisation of its entire genome. The H. polymorpha RB11 genome consists of approximately 9.5 Mb and is organised as six chromosomes ranging in size from 0.9 to 2.2 Mb. Over 90% of the genome was sequenced with concomitant high accuracy and assembled into 48 contigs organised on eight scaffolds (supercontigs). After manual annotation 4767 out of 5933 open reading frames (ORFs) with significant homologies to a non-redundant protein database were predicted. The remaining 1166 ORFs showed no significant similarity to known proteins. The number of ORFs is comparable to that of other sequenced budding yeasts of similar genome size.