MLC-specific suppressor T lymphocytes in man. I. Their induction in vitro by soluble HLA-DR antigens

Human T lymphocytes precultured for 36 hr in the presence of soluble HLA-DR antigens suppress the MLR response of autologous peripheral blood lymphocytes to allogeneic stimulating cells. The suppression is DR antigen-specific in that it appears that the MLR stimulating cell donor and the soluble suppressor-inducing antigen must share DR specificities. The soluble DR antigens were fractionated from the sera of normal donors using QAE-Sephadex chromatography and CNBr-activated Sepharose immunoadsorption. Similarly prepared HLA-A and -B antigens failed to induce suppressive activity. The suppressive activity of DR-antigen cultured T cells is resistant to mitomycin C treatment and, further, the antigen specificity is maintained with or without mitomycin C treatment. The kinetics of suppressor cell induction as well as the kinetics of suppression in the test MLR cultures are presented. The implications of these results are discussed.     

N-(carboxymethylidene)chitosans and N-(carboxymethyl)chitosans: Novel chelating polyampholytes obtained from chitosan glyoxylate

Glyoxylic acid, added to aqueous suspensions of chitosan, causes immediate dissolution of chitosan and gel formation within 3–4 h if the pH is 4.5–5.5. Solutions at lower pH values gel after 2 min of warming at 60–80°. Chitosan glyoxylate solutions brought to alkaline pH with sodium hydroxide do not precipitate chitosan. Evidence is given that a Schiff base, namely N-(carboxymethylidene)chitosan, is formed. N-(Carboxymethylidene)chitosans are reduced by sodium cyanoborohydride at room temperature to give N-(carboxymethyl)chitosans, obtained as white, free-flowing powders, soluble in water at all pH values. A series of N-(carboxymethyl)chitosans having various degrees of acetylation and N-carboxymethylation was obtained, and characterized by viscometry, elemental analysis, and i.r. spectrometry. For the fully substituted N-(carboxymethyl)chitosans, the pK′ is 2.3, the pK″ is 6.6, and the isoelectric point is 4.1. The addition of N-(carboxymethyl)chitosan to solutions (0.2–0.5mm) of transition-metal ions produces immediate insolubilization of N-(carboxymethyl)chitosan-metal ion chelates.     

Plasma membrane association and preliminary characterization of Drosophila sperm surface glycosidases

Previous studies have identified β‐N‐acetylglucosaminidase (GlcNAc'ase) and α‐mannosidase activities on the Drosophila melanogaster sperm surface which may have a role in fertilization. The aim of this study was to investigate their linkage to the sperm plasma membrane. We verified that glycosidases are not peripherally adsorbed to the cell surface by evaluating their resistance to release by KI, by buffered salt solutions of high ionic strength or alkaline buffers. Glycosidases were released from the sperm surface by detergents and, only to a minor extent, by mild proteolysis. Differential detergent solubilization pointed out that Triton X‐114 was the most effective releasing agent for GlcNAc'ase and CHAPS for mannosidase. No activity was released from the membrane by a phosphatidylinositol‐specific phospholipase C (PI‐PLC). The released forms were quite hydrophilic in phase separation experiments with Triton X‐114. This finding indicates the presence of a hydrophobic domain limited to a single transmembrane helix or/and the presence of an extensive glycosilation. The use of a Con‐A binding assay demonstrated that both the enzymes are glycosilated. The molecular weight of the released glycosidases estimated by gel filtration was 158 kDa for GlcNAc'ase and 317 kDa for mannosidase. These results suggest that Drosophila melanogaster GlcNAc'ase and mannosidase are mannosylated integral membrane proteins that would function as exoenzymes with their active sites accessible in the extracellular space. Mol. Reprod. Dev. 52:166–173, 1999. © 1999 Wiley‐Liss, Inc.     

Controlled RISC loading efficiency of miR168 defined by miRNA duplex structure adjusts ARGONAUTE1 homeostasis

Micro RNAs (miRNAs) are processed from precursor RNA molecules with precisely defined secondary stem-loop structures. ARGONAUTE1 (AGO1) is the main executor component of miRNA pathway and its expression is controlled via the auto-regulatory feedback loop activity of miR168 in plants. Previously we have shown that AGO1 loading of miR168 is strongly restricted leading to abundant cytoplasmic accumulation of AGO-unbound miR168. Here, we report, that intrinsic RNA secondary structure of MIR168a precursor not only defines the processing of miR168, but also precisely adjusts AGO1 loading efficiency determining the biologically active subset of miR168 pool. Our results show, that modification of miRNA duplex structure of MIR168a precursor fragment or expression from artificial precursors can alter the finely adjusted loading efficiency of miR168. In dcl1-9 mutant where, except for miR168, production of most miRNAs is severely reduced this mechanism ensures the elimination of unloaded AGO1 proteins via enhanced AGO1 loading of miR168. Based on this data, we propose a new competitive loading mechanism model for miR168 action: the miR168 surplus functions as a molecular buffer for controlled AGO1 loading continuously adjusting the amount of AGO1 protein in accordance with the changing size of the cellular miRNA pool.     

The human CD38 gene: polymorphism, CpG island, and linkage to the CD157 (BST-1) gene

 CD38 is a leukocyte activation antigen and ectoenzyme [NAD(P)⁺ glycohydrolase; EC 3.2.2.6] involved in numerous immune functions. The human CD38 gene is complex [eight exons, >80 kilobases (kb) long] located on Chromosome 4p15, and part of the eukaryotic NAD⁺ glycohydrolase/ADP-ribosyl cyclase gene family. Because of the increasing relevance of the CD38 molecule in the host immune response to infectious, tumoral, and metabolic diseases, we investigated the genetic variability and linkage of the human CD38 locus. We report that (1) the restriction endonuclease Pvu II identifies a bi-allelic polymorphism here defined as formed by the alleles CD38 ^* A (12 kb) and CD38 ^* B (9/2.5 kb); (2) their frequency in the healthy Italian Caucasian population is 14% and 86%, respectively; (3) the polymorphic Pvu II site is located at the 5′ end of the first intron of the CD38 gene; (4) in conjunction with the polymorphic site, we identified a 900 base pair CpG island associated with the CD38 gene, with two potential Sp1 binding sites; (5) the CpG island may play a role in the regulation of CD38 expression and is hypomethylated in various cell lines; (6) by pulsed-field gel electrophoresis we show that CD38 and its paralogue, the bone-marrow stromal cell antigen BST-1 (CD157), map to the same 800 kb Avi II fragment, indicating that the two human ecto-NADase genes are closely linked. Received: 16 December 1998 / Revised: 26 January 1999     

SO2-induced decrease in photosynthetic activity in two barley cultivars. Evidence against specific damage at the protein-pigment complex level

This research investigated the SO₂-induced effects on photosynthetic apparatus in two barley (Hordeum vulgare L.) cultivars (cv), Panda and Express. Following a chronic treatment with SO₂ (80 ppb, 75 d) neither cv showed any visible signs of injury or chlorosis on leaf surfaces, while a significant reduction in A_max and G_w was detected in both cvs, although to different extents. Thylakoids of SO₂-treated plants showed a decrease in the electron transport activity at the whole chain, photosystem II (PSII) and photosystem I (PSI) level in both cvs. The high performance liquid chromatography (HPLC) analysis of leaf pigments revealed a significant decrease in both cvs, more pronounced in Panda than in Express. Deriphat-polyacrilamide gel electrophoresis (Deriphat-PAGE) and two dimensional (2-D) electrophoretic analyses of the pigment-protein complexes did not show differences in SO₂-treated samples of either cv. HPLC analysis of the green bands also showed no differences in the pigment content of fumigated samples of either cv, except for a decrease in β-carotene content and xanthophyll cycle pigment (VAZ) content respectively at band 1 (PSI) and band 5 (minor light-harvesting polypeptides of PSII) level in cv Panda, where the de-epoxidation index (DEP) significantly increased, while in Express, an increase in VAZ content and DEP value of band 5 was observed. These results suggest that the decrease in the photosynthetic activity can be ascribed, in addition to stomata closure induced by SO₂, to a generalised, rather than specific, pollutant effect on photosynthetic apparatus, which could be interpreted as an adaptation to the adverse environment.     

Comparative Analysis of Mammal Genomes Unveils Key Genomic Variability for Human Life Span

The enormous mammal’s lifespan variation is the result of each species’ adaptations to their own biological trade-offs and ecological conditions. Comparative genomics have demonstrated that genomic factors underlying both, species lifespans and longevity of individuals, are in part shared across the tree of life. Here, we compared protein-coding regions across the mammalian phylogeny to detect individual amino acid (AA) changes shared by the most long-lived mammals and genes whose rates of protein evolution correlate with longevity. We discovered a total of 2,737 AA in 2,004 genes that distinguish long- and short-lived mammals, significantly more than expected by chance (P = 0.003). These genes belong to pathways involved in regulating lifespan, such as inflammatory response and hemostasis. Among them, a total 1,157 AA showed a significant association with maximum lifespan in a phylogenetic test. Interestingly, most of the detected AA positions do not vary in extant human populations (81.2%) or have allele frequencies below 1% (99.78%). Consequently, almost none of these putatively important variants could have been detected by genome-wide association studies, suggesting that comparative genomics can be used to complement and enhance interpretation of human genome-wide association studies. Additionally, we identified four more genes whose rate of protein evolution correlated with longevity in mammals. Finally, we show that the human longevity-associated proteins are significantly more stable than the orthologous proteins from short-lived mammals, strongly suggesting that general protein stability is linked to increased lifespan.     

Large-scale production of peptides using the solid phase continuous flow method. Part 2: preparative synthesis of a 26-mer peptide thrombin inhibitor

A preparative method for the preparation of large peptides is described. An advantageous theoretical weight of peptide/weight of starting resin ratio (tP_w/R_w) of about 0.3 was successfully experimented. The esterification of the first amino acid was realized with a racemization of less than 1%. The study of the coupling conditions led to the use of a diluted acylating mixture that allowed a 56% consumption of the amino acid derivatives (percentage use of amino acids) introduced in the synthesis. The cost analysis of the synthesis showed that the recovery of the amino acid derivatives was not worthwhile. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

DNA barcoding of marine fishes from Saudi Arabian waters of the Gulf

We used the cytochrome oxidase subunit I (coI) gene DNA to barcode 117 endemic Gulf and cosmopolitan Indo–West Pacific fish species belonging to 54 families and 13 orders. Novel DNA barcodes were provided for 18 fish species (Trachinocephalus sp., Nematalosa sp., Herklotsichthys lossei, Upeneus doriae, Trachurus indicus, Apogonichthyoides taeniatus, Verulux cypselurus, Favonigobius sp., Suezichthus gracilis, Sillago sp., Brachirus orientalis, Pegusa sp., Lepidotrigla bispinosa, Lepidotrigla sp., Grammoplites suppositus, Hippichthys sp., Paramonacanthus sp. and Triacanthus sp.). The species delimitation analysis, conducted with Poisson tree processes– Bayesian PTP (PTP–bPTP) and nucleotide-divergence-threshold (NDT) models), found 137 and 119 entities respectively. Overall, NDT method, neighbour-joining species tree and the prior taxonomic assessment provided similar results. Among the 54 families considered, only 10 (Ariommatidae, Ephippidae, Leiognathidae, Nemipteridae, Plotosidae, Pomacanthidae, Pomacentridae, Priacanthidae and Rachycentridae) showed the occurrence of molecular diagnostic pure characters. The DNA barcoding database developed during this study will help ichthyologists to identify and resolve the taxonomic ambiguities they may encounter with the fishes occurring in The Gulf and throughout the region.     

Assessment of the Chemical Quality of Recreational Bathing Water in Argentina by Health Risk Analysis

Del Azul creek (Argentina) is a natural water body used for recreational bathing in which heavy metals and pesticides have been detected. The aim of the study is to estimate the probabilistic non-cancer and cancer risks by recreational bathing, applying U.S. Environmental Protection Agency models for aggregated (exposure through accidental oral intake of water and dermal contact) and cumulative risks (combined exposure to groups of substances) for bathers of 5, 10, 15, and 20 years old. Bathing in Del Azul creek does not generate risks at the concentrations and the exposure scenarios considered. Cypermethrin and arsenic and heptachlor were the riskiest non-cancer and cancer substances, respectively. Our study highlights the importance of considering both routes of exposure because of the great significance of the dermal route and because of the variability of population characteristics, as it has been stated in other studies. These considerations are highly significant for Argentina, where the quality of recreational water for other than microbiological causes is frequently evaluated based only on the harmful oral intake and applied to a hypothetical individual representative of the population.