A study of the selective inhibition by urea of neutrophil alkaline phosphatase isoenzymes

The kinetics of neutrophil alkaline phosphatase (AP) inactivation by urea were examined. In standardized conditions (molarity and pH of buffer, magnesium chloride and urea concentration), monitoring of the enzymatic reaction allows identification of neutrophil AP isoenzymes. This study confirms the heterogeneity of AP in normal human neutrophils.     

Enzyme polymorphisms of Ideles populations (Ahaggar, Algeria) and the Iwellemeden Kel Kummer Twaregs (Menaka, Mali).

Surveys dealing with enzyme polymorphisms have recently been conducted in the Sahara. Results from two populations are reported here: 227 inhabitants of Ideles village (Ahaggar, Algeria); 285 nomads of a genetic isolate, the Kel Kummer Twareg tribe (Menaka, Mali). The four classical molecular variants of G6PD:A+, A-, B+, B-, are found in Ideles. The frequency of the G6PD A+ Negroid variant reaches 15% in Ideles and 7.7% among the Kel Kummer. However, gene frequencies will have to be recalculated after a study of the genetic transmission through families. The PGDC gene of 6PGD is especially frequent in the Kel Kummer where 10 'Canning' phenotypes have been observed. The PGM distribution of alleles at locus 1 in Ideles is the same as in the Mediterranean populations. The pa gene of acid phosphatase, relatively frequent in Ideles, has been excluded by drift from the Kel Kummer gene pool. AK and LDH enzymes have also been studied in both samples. The abnormal Ea1 mutation of serum pseudocholinesterase exists in Ideles and in the Kel Kummer as in other populations of the Sahara; the C5 esterase component was revealed by electrophoresis in 5% of the Kel Kummer people.     

Alkaline phosphatase and phosphotyrosine phosphatase activities of cultured amniotic cells with trisomy 18.

In cultured amniotic cells from fetuses with Edward's syndrome (trisomy 18), the activities of two protein phosphatases, alkaline phosphatase and phosphotyrosine phosphatase, were measured. Comparison with normal fetal cells showed a different behavior for each enzyme. Alkaline phosphatase was significantly lowered while phosphotyrosine phosphatase remained at normal levels. The interest of these enzyme assays in the screening procedure of this severe chromosome defect is discussed.     

cDNA to chick lumican (corneal keratan sulfate proteoglycan) reveals homology to the small interstitial proteoglycan gene family and expression in muscle and intestine.

A 1.9-kb cDNA clone to chick lumican (keratan sulfate proteoglycan) was isolated by screening an expressing vector library made from chick corneal RNA with antiserum to chick corneal lumican. The cDNA clone contained an open reading frame coding for a 343-amino acid protein, Mr = 38,640. Structural features of the deduced sequence include: a 18-amino acid signal peptide, cysteine residues at the N- and C-terminal regions, and a central leucine-rich region (comprising 62% of the protein) containing nine repeats of the sequence LXXLXLXXNXL/I, where X represents any amino acid. Lumican contains three variations of this sequence that are tandemly linked to form a unit and three units tandemly linked to form the leucine-rich region. The sequential arrangement of these repeats and their spacing suggest that this region arose by duplication. The deduced sequence shows five potential N-linked glycosylation sites, four of which are in the leucine-rich region. These sites are also potential keratan sulfate attachment sites. The cDNA clone to lumican hybridizes to a 2.0-kb mRNA found in tissues other than cornea, predominantly muscle and intestine. Radiolabeling and immunoprecipitation studies show that lumican core protein is also synthesized by these tissues. The primary structure of lumican is similar to fibromodulin, decorin, and biglycan, which indicates it belongs to the small interstitial proteoglycan gene family. The expression of lumican in tissues other than cornea indicates a broader role for lumican besides contributing to corneal transparency.     

Properties of a basement membrane-related glycoprotein synthesized in culture by a mouse embryonal carcinoma-derived cell line.

Two glycoproteins, GP-1 and GP-2, have been isolated from an extracellular membrane synthesized in cell culture by an embryonal carcinoma-derived cell line. The amino acid and carbohydrate compositions have been determined. Both proteins are rich in half-cystine residues and contain approximately 12-15% carbohydrate. Antibodies have been obtained against one of the glycoproteins, GP-2, in rabbits. The antibody reacts with basement membranes from adult mouse and human kidney glomeruli and tubules, and all basement membranes tested from mouse embryonic tissues. The molecular properties of GP-2 are superficially similar to LETS protein; however, immunological and other criteria show that they are distinct proteins. The presence of LETS protein and GP-2 in basement membranes suggests that there are subtle interactions which are important in adhesion of epithelial cells to basement membranes.     

Erythrocyte glucose-6-phosphate dehydrogenase in the Niokolonko (Malinke of the Niokolo) of Eastern Senegal. Identification of a slow variant with normal activity (Tacoma-like).

In a survey of blood genetic markers in the Niokolonko of Eastern Senegal, three types of G6PD variants were discovered: (1) fast variants, common Negro G6PD A +; the frequency of the Gd A + gene was 0.183; (2) deficient G6PD A--, occurring with a fairly low frequency: 0.079, and (3) some individuals were carriers of a slow moving electrophoretic variant with normal activity. After purification, the analysis of kinetic parameters showed that this enzyme was closely similar to G6PD Tacoma. We proposed to label it 'G6PD Tacoma-like'. The incidence of this mutation in the whole group studied was 0.020. G6PD Tacoma-like may be common in some African tribes.     

An experimental system to study responses of Medicago truncatula roots to chitin oligomers of high degree of polymerization and other microbial elicitors

Key message

A fully acetylated, soluble CO preparation of mean DP of ca. 7 was perceived with high sensitivity by M. truncatula in a newly designed versatile root elicitation assay.

Abstract

The root system of legume plants interacts with a large variety of microorganisms, either pathogenic or symbiotic. Understanding how legumes recognize and respond specifically to pathogen-associated or symbiotic signals requires the development of standardized bioassays using well-defined preparations of the corresponding signals. Here we describe the preparation of chitin oligosaccharide (CO) fractions from commercial chitin and their characterization by a combination of liquid-state and solid-state nuclear magnetic resonance spectroscopy. We show that the CO fraction with highest degree of polymerization (DP) became essentially insoluble after lyophilization. However, a fully soluble, fully acetylated fraction with a mean DP of ca. 7 was recovered and validated by showing its CERK1-dependent activity in Arabidopsis thaliana. In parallel, we developed a versatile root elicitation bioassay in the model legume Medicago truncatula, using a hydroponic culture system and the Phytophthora β-glucan elicitor as a control elicitor. We then showed that M. truncatula responded with high sensitivity to the CO elicitor, which caused the production of extracellular reactive oxygen species and the transient induction of a variety of defense-associated genes. In addition, the bioassay allowed detection of elicitor activity in culture filtrates of the oomycete Aphanomyces euteiches, opening the way to the analysis of recognition of this important legume root pathogen by M. truncatula.     

Cholesterol and cholate components of an atherogenic diet induce distinct stages of hepatic inflammatory gene expression

Atherosclerosis in inbred mouse strains has been widely studied by using an atherogenic (Ath) diet containing cholesterol, cholic acid, and fat, but the effect of these components on gene expression has not been systematically examined. We employed DNA microarrays to interrogate gene expression levels in liver of C57BL/6J mice fed the following five diets: mouse chow, the Ath diet, or modified versions of the Ath diet in which either cholesterol, cholate, or fat were omitted. Dietary cholesterol and cholate produced discrete gene expression patterns. Cholesterol was required for induction of genes involved in acute inflammation, including three genes of the serum amyloid A family, three major histocompatibility class II antigen genes, and various cytokine-related genes. In contrast, cholate induced expression of genes involved in extracellular matrix deposition in hepatic fibrosis, including five collagen family members, collagen-interacting proteins, and connective tissue growth factor. The gene expression findings were confirmed by biochemical measurements showing that cholesterol was required for elevation of circulating serum amyloid A, and cholate was required for accumulation of collagen in the liver. The possibility that these gene expression changes are relevant to atherogenesis in C57BL/6J mice was supported by the observation that the closely related, yet atherosclerosis-resistant, C57BL/6ByJ strain was largely resistant to dietary induction of the inflammatory and fibrotic response genes. These results establish that cholesterol and cholate components of the Ath diet have distinct proatherogenic effects on gene expression and suggest a strategy to study the contribution of acute inflammatory response and fibrogenesis independently through dietary manipulation.     

Cytoplasmic SIR2 homologue overexpression promotes survival of Leishmania parasites by preventing programmed cell death

The Silent Information Regulator (SIR2) family of genes have been cloned from a variety of species ranging from bacteria to man. In previous studies, we reported the characterization of a Leishmania major gene encoding a protein with extensive homology to yeast SIR2p and expressed by different Leishmania species and parasite developmental stages and thus termed LmSIR2. Unlike the yeast SIR2p, LmSIR2p is mainly localized within the cytoplasm. In the present study, sequencing of a homologue encoding gene in another Leishmania species, Leishmania infantum, revealed 93% overall amino acid identity with L. major SIR2 gene. Further, using L. infantum as a recipient for a plasmid vector (pTEX) which allows overexpression of LmSIR2p led to the accumulation of the protein in the parasite cytoplasm of both promastigote and amastigote forms and a striking increase in the survival of amastigotes, the vertebrate stage of the parasite, when maintained under normal axenic culture conditions. This phenotype was also observed when L. infantum parasites were transfected with a cosmid vector (CLHyg), isolated from a L. infantum cosmid library, carrying the L. infantum SIR2 gene (CLHyg-LiSIR2). In contrast, no effect was observed on survival of the promastigote forms (insect stage) under similar culture conditions. However, when the glucose was used as a unique source of energy under starvation conditions, the viability of promastigotes was significantly enhanced. Moreover, we showed that amastigote forms in the stationary phase of culture died with a feature of apoptosis as revealed by the appearance of YOPRO-1 positive cells and that expression of LmSIR2 protein substantially delays this phenomenon. Taken together, these results demonstrate the existence of SIR2-related proteins encoding genes in different Leishmania species and suggest that LmSIR2p could participate among other factors in the control of cell death.