Changes in surface glycopeptides after malignant transformation of rat liver cells and during the regression of hepatoma cells

Normal liver cells, Zajdela's hepatoma cells, and regressing hepatoma cells were metabolically labeled with either radioactive glucosamine or mannose. Glycopeptides obtained by exhaustive pronase digestion of these cells were compared after fractionation by gel filtration on Bio-Gel P-6. Chemical analysis, affinity chromatography on immobilized lectins, alkaline treatment, and susceptibility toward endo-beta-N-acetylglucosaminidase and tunicamycin revealed dramatic changes in the glycopeptide patterns of transformed cells during the recovery of normal phenotype. The most prominent feature was the presence on the surface of hepatoma cells of a large glycopeptide, which was absent from normal liver cells and disappeared almost completely during the regression of hepatoma cells. This large glycopeptide had a Mr of 70,000, contained essentially O-glycosidically linked glycan chains, and did not result from a hypersialylation. N-glycosidically linked glycopeptides, high-mannose, and complex-type oligosaccharides were present in distinct proportions according to the differentiation state. Transformation of liver cells led to a reduction of high-mannose type oligosaccharides and an increase in the degree of branching of complex-type oligosaccharides. In addition, "bisected" glycopeptides were present only on hepatoma cells. The pattern of N-linked glycopeptides of normal liver cells was recovered during the regression of hepatoma cells. The origin of glycopeptide differences between normal and transformed cells and the evidence of a relation between carbohydrate changes, in particular the appearance of a large glycopeptide, and tumorigenicity are discussed.     

Three bovine alpha2-fucosyltransferase genes encode enzymes that preferentially transfer fucose on Galbeta1-3GalNAc acceptor substrates

To investigate the synthesis of alpha2-fucosylated epitopes in the bovine species, we have characterized cDNAs from various tissues. We found three distinct alpha2-fucosyltransferase genes, named bovine fut1, fut2, and sec1 which are homologous to human FUT1, FUT2, and Sec1 genes, respectively. Their open reading frames (ORF) encode polypeptides of 360 (bovine H), 344 (bovine Se), and 368 (bovine Sec1) amino acids, respectively. These enzymes transfer fucose in alpha1,2 linkage to ganglioside GM(1)and galacto- N -biose, but not to the phenyl-beta-D-galactoside, type 1 or type 2 acceptors, suggesting that their substrate specificity is different and more restricted than the other cloned mammalian alpha2-fucosyltransferases. Southern blot analyses detected four related alpha2-fucosyltransferase sequences in the bovine genome while only three have been described in other species. The supernumerary entity seems to be related to the alpha2-fucosyltransferase activity which can also use type 1 and phenyl-beta-D-galactoside substrate acceptors. It was exclusively found in bovine intestinal tract. Our results show that, at least in one mammalian species, four alpha2-fucosyltransferases are present, three adding a fucose on alpha1,2 linkage on type 3/4 acceptor (Galbeta1-3GalNAc) and another able to transfer also fucose on phenyl-beta-D-galactoside and type 1 (Galbeta1-3GlcNAc) acceptors. The phylogenetic tree of the enzymes homologous to those encoded by the bovine fut1, fut2, and sec1 genes revealed two main families, one containing all the H-like proteins and the second containing all the Se-like and Sec1-like proteins. The Sec1-like family had a higher evolutionary rate than the Se-like family.     

Development and validation of PCR primers to assess the diversity of Clostridium spp. in cheese by temporal temperature gradient gel electrophoresis

A nested-PCR temporal temperature gradient gel electrophoresis (TTGE) approach was developed for the detection of bacteria belonging to phylogenetic cluster I of the genus Clostridium (the largest clostridial group, which represents 25% of the currently cultured clostridial species) in cheese suspected of late blowing. Primers were designed based on the 16S rRNA gene sequence, and the specificity was confirmed in PCRs performed with DNAs from cluster I and non-cluster I species as the templates. TTGE profiles of the PCR products, comprising the V5-V6 region of the 16S rRNA gene, allowed us to distinguish the majority of cluster I species. PCR-TTGE was applied to analyze commercial cheeses with defects. All cheeses gave a signal after nested PCR, and on the basis of band comigration with TTGE profiles of reference strains, all the bands could be assigned to a clostridial species. The direct identification of Clostridium spp. was confirmed by sequencing of excised bands. C. tyrobutyricum and C. beijerinckii contaminated 15 and 14 of the 20 cheese samples tested, respectively, and C. butyricum and C. sporogenes were detected in one cheese sample. Most-probable-number counts and volatile fatty acid were determined for comparison purposes. Results obtained were in agreement, but only two species, C. tyrobutyricum and C. sporogenes, could be isolated by the plating method. In all cheeses with a high amount of butyric acid (>100 mg/100 g), the presence of C. tyrobutyricum DNA was confirmed by PCR-TTGE, suggesting the involvement of this species in butyric acid fermentation. These results demonstrated the efficacy of the PCR-TTGE method to identify Clostridium in cheeses. The sensitivity of the method was estimated to be 100 CFU/g.     

Solution NMR structure of the SH3 domain of human nephrocystin and analysis of a mutation-causing juvenile nephronophthisis

Human nephrocystin is a protein associated with juvenile NPH, an autosomal recessive, inherited kidney disease responsible for chronic renal failure in children. It contains an SH3 domain involved in signaling pathways controlling cell adhesion and cytoskeleton organization. The solution structure of this domain was solved by triple resonance NMR spectroscopy. Within the core, the structure is similar to those previously reported for other SH3 domains but exhibits a number of specific noncanonical features within the polyproline ligand binding site. Some of the key conserved residues are missing, and the N-Src loop exhibits an unusual twisted geometry, which results in a narrowing of the binding groove. This is induced by the replacement of a conserved Asp, Asn, or Glu residue by a Pro at one side of the N-Src loop. A systematic survey of other SH3 domains also containing a Pro at this position reveals that most of them belong to proteins involved in cell adhesion or motility. A variant of this domain, which carries a point mutation causing NPH, was also analyzed. This change, L180P, although it corresponds to a nonconserved and solvent-exposed position, causes a complete loss of the tertiary structure. Similar effects are also observed with the L180A variant. This could be a context-dependent effect resulting from an interaction between neighboring charged side-chains.     

Organization of the bovine alpha 2-fucosyltransferase gene cluster suggests that the Sec1 gene might have been shaped through a nonautonomous L1-retrotransposition event within the same locus

By referring to the split coding sequence of the highly conserved alpha 6-fucosyltransferase gene family (assumed to be representative of the common alpha 2 and alpha 6 fucosyltransferase gene ancestor), we have hypothesized that the monoexonic coding sequences of the present alpha 2-fucosyltransferase genes have been shaped in mammals by several events of retrotransposition and/or duplication. In order to test our hypothesis, we determined the structure of the three bovine alpha 2-fucosyltransferase genes (bfut1, bfut2, and sec1) and analyzed their characteristics compared with their human counterparts (FUT1, FUT2, and Sec1). We show that in mammals, a complex nonautonomous L1-retrotransposition event occurred within the locus of the alpha 2-fucosyltransferase ancestor gene itself. A consequence of this event was the processing in Catarrhini of a Sec1 pseudogene via several point mutations.     

Effect of moderate hypoxemia on atrial natriuretic factor and arginine vasopressin in normal man

The object of this study was to assess the effect of moderate acute hypoxemia on plasma concentrations of atrial natriuretic factor (ANF), arginine vasopressin (AVP), plasma renin activity (PRA) and urinary excretion of prostaglandin E2 (UPGE2V). Eight volunteers were exposed for 2 hours to a gas mixture containing 10% O2, 4.5% CO2 and 85.5% N2. Hypoxia increased diastolic blood pressure and free water clearance. Hypoxia did not change the AVP, PRA or UPG2V, although increased ANF from 17.7 +/- 3.4 pg/mL to 27.2 +/- 1.7 pg/mL (p less than 0.005) at 120 minutes. ANF changes were closely associated with the rise in blood pressure.     

Integrity of the Saccharomyces cerevisiae Rpn11 Protein Is Critical for Formation of Proteasome Storage Granules (PSG) and Survival in Stationary Phase

Decline of proteasome activity has been reported in mammals, flies and yeasts during aging. In the yeast Saccharomyces cerevisiae, the reduction of proteolysis in stationary phase is correlated with disassembly of the 26S proteasomes into their 20S and 19S subcomplexes. However a recent report showed that upon entry into the stationary phase, proteasome subunits massively re-localize from the nucleus into mobile cytoplasmic structures called proteasome storage granules (PSGs). Whether proteasome subunits in PSG are assembled into active complexes remains an open question that we addressed in the present study. We showed that a particular mutant of the RPN11 gene (rpn11-m1), encoding a proteasome lid subunit already known to exhibit proteasome assembly/stability defect in vitro, is unable to form PSGs and displays a reduced viability in stationary phase. Full restoration of long-term survival and PSG formation in rpn11-m1 cells can be achieved by the expression in trans of the last 45 amino acids of the C-terminal domain of Rpn11, which was moreover found to co-localize with PSGs. In addition, another rpn11 mutant leading to seven amino acids change in the Rpn11 C-terminal domain, which exhibits assembled-26S proteasomes, is able to form PSGs but with a delay compared to the wild type situation. Altogether, our findings indicate that PSGs are formed of fully assembled 26S proteasomes and suggest a critical role for the Rpn11 protein in this process.