Laminin affects polymerization,depolymerization and neurotoxicity of Abeta peptide

Amyloid deposition in Alzheimer fibrils forms neurotoxic senile plaques in a process that may be modulated by associated proteins. In this work we demonstrate the ability of laminin-1 and laminin-2 to inhibit fibril formation and toxicity on cultured rat hippocampal neurons. We confirm that the laminin-1-derived peptide YFQRYLI inhibits efficiently both fibril formation and neurotoxicity and show that the IKVAV peptide inhibits amyloid neurotoxicity despite its slight inhibition of fibril formation. On other hand, laminin-1 induces disaggregation of preformed fibrils in vitro, characterized as a progressive disassembly of fibrils into protofibrils and further clearance of these latter species, leading to a continual inhibition of amyloid neurotoxicity.     

Wnt signaling involvement in beta-amyloid-dependent neurodegeneration

Alzheimer's disease (AD) is a progressive dementia paralleled by selective neuronal death, which is probably caused by the cytotoxic effects of the amyloid-beta peptide (Abeta). We have observed that Abeta-dependent neurotoxicity induces a loss of function of Wnt signaling components and that activation of this signaling cascade prevent such cytotoxic effects. Therefore we propose that compounds which mimic this signaling cascade may be candidates for therapeutic intervention in Alzheimer's patients.     

Immunohistochemical analysis of beta3 integrin (CD61): expression in pig tissues and human tumors

CD61 is a membrane glycoprotein that associates with CD41 (alphaIIb) to form the heterodimeric complex gpIIb/IIIa (CD41/CD61), predominantly expressed in platelets and megakariocytes. CD61 or beta3 integrin also associates with alpha v (CD51) to form the vitronectin receptor, which is expressed in many tissues. We have used a monoclonal antibody against the porcine gpIIIa or CD61 (JM2E5) to study the distribution of this molecule in different normal pig tissues. As in humans, CD61 was broadly expressed in all tissues examined. In the kidney, strong expression of CD61 was observed in epithelial cells from renal tubules. In the testis, CD61 expression was detected in the Leydig cells. However, in liver, CD61 was weak or not detected. Many integrins are particularly involved in tumogenicity and in tumor progression mediating cell-cell interaction. Immunofluorescence experiments using cultured human tumor HeLa cells showed nuclear and cytoplasmic staining of mAb JM2E5. Immunohistochemical analysis of human tumor sections from several organs showed a heterogeneus distribution in metastatic cases from colon and breast carcinoma. However, no staining was found in metastasis from melanoma.     

Multiple mechanisms generate HLA class I altered phenotypes in laryngeal carcinomas: high frequency of HLA haplotype loss associated with loss of heterozygosity in chromosome region 6p21

Major histocompatibility complex (MHC) class I loss or downregulation in cancer cells is a major immune escape route used by a large variety of human tumors to evade anti-tumor immune responses mediated by cytotoxic T lymphocytes. Multiple mechanisms are responsible for such HLA class I alterations. However, the precise frequency of these molecular defects has not been clearly determined in tumors derived from specific tissues. To analyze such defects we aim to define the major HLA class I-altered phenotypes in different tumor types. In this paper we report on HLA class I expression in 70 laryngeal carcinomas. We used immunohistological techniques with a highly selective panel of anti-HLA monoclonal antibodies (mAb), and polymerase chain reaction (PCR) microsatellite amplification of previously selected microsatellite markers (STR) located in chromosome 6 and 15. DNA was obtained from microdissected tumor tissues and surrounding stroma to define the loss of heterozygosity (LOH) associated with chromosome 6p21. Our results showed that LOH in chromosome 6 produced HLA haplotype loss (phenotype II) in 36% of the tumors. In addition, HLA class I total loss (phenotype I) was found in 11%; HLA A or B locus downregulation (phenotype III) was detected in 20%; and HLA class I allelic loss (phenotype IV) in 10% of all cases. We sometimes observed two or more associated mechanisms in the same HLA-altered phenotype, such as LOH and HLA total loss in phenotype I. In only 23% of tumors it was not possible to identify any HLA class I alteration. We conclude that the combination of immunohistological techniques and molecular analysis of tumor DNA obtained from microdissected tumor tissues provides a means for the first time of determining the actual frequency of the major HLA class I-altered phenotypes in laryngeal carcinomas.     

Effects of 15N application frequency on nitrogen uptake efficiency in citrus trees

Two irrigation systems were used to compare nitrogen uptake efficiency in citrus trees and to evaluate the NO₃⁻ runoff in «Navelina» orange trees [Citrus sinensis (L.) Osbeck] on Carrizo citrange rootstock (Citrus sinensis × Poncirus trifoliata Raf.). These were fertilized with 125 g N as labelled K¹⁵NO₃ and grown outdoors in containers filled with a sand-loamy soil. Two groups of 3 trees received this N dose either in five equally split applications by a flooding irrigation system or in 66 applications by drip. Trees were harvested at the end of the vegetative cycle (December) and the isotopic ratios of ¹⁵N/¹⁴N were measured in the soil-plant system. The N uptake efficiency of the whole tree was higher with drip irrigation (75 percnt;) than with flooding system (64 percnt;). In the 0-90 cm soil profile, the N immobilized in the organic fraction was similar for both irrigation methods (around 13 percnt;), whereas the N retained as NO₃⁻ was 1 percnt; of the N applied under drip and 10 percnt; under flooding. In the last case, most of NO₃⁻ remained under root system and it could be lost to leaching either by heavy rainfalls or excessive water applications. These results showed that a drip irrigation system was more efficient for improving water use and N uptake from fertilizer, in addition to potentially reduced leaching losses.     

Three-color versus four-color multiparameter cell cycle analyses of primary acute myeloid leukemia samples

Checkpoint alterations that impact cell cycle and apoptosis responses to therapeutic treatments may produce drug resistance in acute myeloid leukemia (AML). To study these, we have developed flow cytometry assays of checkpoint function that also allow quantitation of key molecular regulators of apoptosis and cell cycle. We have used three-color (3C) assays, with FITC-labeled anti-BCL-2 and PE-labeled anti-proliferating cell nuclear antigen (PCNA) antibodies, and the DNA dye 7-aminoactinomycin, to characterize primary leukemia cells identified in DNA x side light scatter (SSC) histograms. We showed that 3C assays are accurate and reproducible in analyses of leukemia cell lines and of primary AML and normal bone marrow samples (Banker et al.: Blood 89: 243-255, 1997; Banker et al.: Leukemia Res 22: 221-239, 1998; Banker et al.: Clin Cancer Res 4: 3051-3062, 1998). To further confirm the validity of our SSC leukemia cell gating and to address whether immunophenotypic AML subsets might have different biologic properties, we have now designed four-color (4C) flow assays to characterize checkpoint status in leukemic blasts specifically identified by surface immunostaining. In modeling this assay strategy, PE/Cy5-labeled anti-CD34 antibody was used to detect blasts, with FITC-labeled anti-BCL-2, PE-labeled anti-PCNA antibodies, and Hoechst 33342 (H33342) DNA dye. Four-color CD34-gated data was concordant with 3C, SSC-gated data for leukemia cell lines and for most primary AML samples with high and intermediate blast counts. BCL-2 and PCNA immunopositivity and sub-G1 apoptosis determinations were different in the CD34-gated versus SSC-gated blasts in particular samples with smaller CD34(+) subsets, suggesting that leukemia samples can contain blast subsets with different biologic properties. On the other hand, PCNA-gated cell-cycle distributions in untreated cells and G1 versus S phase cell-cycle arrests after cytosine arabinoside treatments were completely concordant in 4C and 3C assays. We conclude that both 3C and 4C assays can be used to characterize protein expression and cell-cycle drug response patterns in leukemia blasts, but that 4C assays may additionally allow discrimination of these properties in immunophenotypic leukemia subsets.     

The crystal structure of tetrameric methionine adenosyltransferase from rat liver reveals the methionine-binding site

Most of the transmethylation reactions use the same methyl donor, S-adenosylmethionine (SAM), that is synthesised from methionine and ATP by methionine adenosyltransferase (MAT). In mammals, two MAT enzymes have been detected, one ubiquitous and another liver specific. The liver enzyme exists in two oligomeric forms, a tetramer (MAT I) and a dimer (MAT III), MAT I being the one that shows a higher level of affinity for methionine but a lower SAM synthesis capacity. We have solved the crystal structure of rat liver MAT I at 2.7 A resolution, complexed with a methionine analogue: l-2-amino-4-methoxy-cis-but-3-enoic acid (l-cisAMB). The enzyme consists of four identical subunits arranged in two tight dimers that are related by crystallographic 2-fold symmetry. The crystal structure shows the positions of the relevant cysteine residues in the chain, and that Cys35 and Cys61 are perfectly oriented for forming a disulphide link. This result leads us to propose a hypothesis to explain the control of MAT I/III exchange and hence, the effects observed on activity. We have identified the methionine-binding site into the active-site cavity, for the first time. The l-cisAMB inhibitor is stacked against Phe251 aromatic ring in a rather planar conformation, and its carboxylate group coordinates a Mg(2+), which, in turn, is linked to Asp180. The essential role of the involved residues in MAT activity has been confirmed by site-directed mutagenesis. Phe251 is exposed to solvent and is located in the beginning of the flexible loop Phe251-Ala260 that is connecting the N-terminal domain to the central domain. We postulate that a conformational change may take place during the enzymatic reaction and this is possibly the reason of the unusual two-step mechanism involving tripolyphosphate hydrolysis. Other important mechanistic implications are discussed on the light of the results. Moreover, the critical role that certain residues identified in this study may have in methionine recognition opens further possibilities for rational drug design.     

Assessment of a monoclonal antibody-based competitive enzyme linked immunosorbent assay (cELISA) for diagnosis of brucellosis in infected and Rev. 1 vaccinated sheep and goats

In this study a cELISA for the diagnosis of brucellosis due to B. melitensis in sheep and goats was evaluated and its capability of discriminating vaccinated from infected animals was assessed. Information is provided indicating that the cELISA has a diagnostic sensitivity (99.4%) and specificity (98.9%) in sheep and goats comparable to that of many standard indirect ELISA methods. In addition, the test proved able to distinguish between vaccinated and infected animals with an accuracy of up to 90% and results reproducibility of 93%. It was concluded that the cELISA could be useful for differentiation of Rev.1 vaccinated and naturally infected sheep and goats.