Block copolymer-oligonucleotide conjugates for genotyping on microarrays

Polymer-oligonucleotide conjugates were synthesized from the amphiphilic block copolymer poly(tert-butylacrylamide-b-(N-acryloylmorpholine-co-N-acryloxysuccinimide)) using an original solid-phase DNA synthesis strategy. This method provided conjugates highly functionalized with oligonucleotides throughout the polymer chain. After purification, block copolymer-oligonucleotide conjugates were spotted on a multidetection microarray system developed by Apibio using a standard nanodroplet piezo inkjet spotting technique to develop the oligosorbent assay (OLISA). Two genotyping models (HLA-DQB1 and platelet glycoproteins [GPs]), which are particularly difficult to study with standard systems, were evaluated. For both models, block copolymer-oligonucleotide conjugates used as capture probes amplified the responses of in vitro diagnostic assays. The detection limit reached by using conjugates was estimated at 15 pM for a 219-bp DNA target (HLA-DQB1 model). Moreover, single nucleotide polymorphism was detected in the platelet GPs genotyping model. The use of polymer conjugates led to a significant improvement in both sensitivity and specificity of standard hybridization assays even when applied to complex biological models.     

Characterization of cellobiohydrolase I (Cel7A) glycoforms from extracts of Trichoderma reesei using capillary isoelectric focusing and electrospray mass spectrometry

Capillary isoelectric focusing (CIEF) was used to profile the cellulase composition in complex fermentation samples of secreted proteins from Trichoderma reesei. The enzyme cellobiohydrolase I (CBH I, also referred to as Cel7A), a major component in these extracts, was purified from different strains and characterized using analytical methods such as CIEF, high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC–PAD), and capillary liquid chromatography–electrospray mass spectrometry (cLC–ESMS). ESMS was also used to monitor the extent of glycosylation in CBH I isolated from T. reesei strain RUT-C30 and two derivative mutant strains. Selective identification of tryptic N-linked glycopeptides was achieved using LC–ESMS on a quadrupole/time-of-flight instrument with a mixed scan function. The suspected glycopeptides were further analyzed by on-line tandem mass spectrometry to determine the nature of N-linked glycans and their attachment sites. This strategy enabled the identification of a high mannose glycan attached to Asn270 (predominantly Man₈GlcNAc₂) and single GlcNAc occupancy at Asn45 and Asn384 with some site heterogeneity depending on strains and fermentation conditions. The linker region of CBH I was shown to be extensively glycosylated with di-, and tri-saccharides at Thr and Ser residues as indicated by MALDI-TOF and HPAEC–PAD experiments. Additional heterogeneity was noted in the CBH I linker peptide of RUT-C30 strain with the presence of a phosphorylated di-saccharide.     

Release of ferulic acid from agroindustrial by-products by the cell wall-degrading enzymes produced by Aspergillus niger I-1472

Aspergillus niger I-1472 was grown on sugar beet pulp to produce cell wall polysaccharide-degrading enzymes, including feruloyl esterases. Compared to enzymatic activities measured in commercially available mixtures previously used for the release of ferulic acid, the A. niger enzymes were more various. These enzymes were tested to release ferulic acid from sugar beet pulp, maize bran, or autoclaved maize bran. They were as efficient as the commercial mixture to release ferulic acid from sugar beet pulp. On the other hand, they were much more efficient to release ferulic acid from maize bran after autoclaving pretreatment, as 95% of ferulic acid ester were solubilized. Thus, A. niger enzymes exhibited a high interest in the release of ferulic acid from various agro-industrial by-products.     

In vitro culture of bovine egg fertilized either in vivo or in vitro

Three-quarters of in vivo and one-third of in vitro fertilized bovine eggs reached blastocyst stage when cultured on tubal cell monolayers (TCM), but no hatching occurred in B2 medium supplemented with estrous cow serum. When after 3 days of culture on TCM, morulae were transferred on endometrial cell monolayers (UCM), the same proportion of blastocysts was obtained and one-third of them hatched. Histological studies of hatched blastocysts showed that the number of inner cells was significantly lower than in hatched blastocysts recovered in vivo 8-8.5 days after ovulation. Moreover, the number of pycnotic cells was higher than normal, although mitosis were present. On the contrary, there was no difference in either the number or the appearance of trophoblastic cells between blastocysts obtained in vitro and in vivo. The addition of transforming growth factor (TGF-beta) to either TCM or UCM co-cultures at the very beginning of blastocyst formation specifically stimulated growth of the inner cell mass (ICM). The number of cells at hatching was about double (120) and significantly higher than that found in 8-8.5-day blastocysts in vivo. Moreover, hatching percentages were similar to the controls, even when eggs were cultured for 8 days only on TCM. However the proportion of pycnotic cells remained higher than normal, although many mitotic cells were unevenly distributed in ICM) In vivo during hatching, there were always pycnotic cells in ICM, but their number was limited and approximately similar to the number of mitosis. The uterine factors which control both mitosis and pycnosis in ICM remain to be discovered.     

Rat pheochromocytoma tyrosine hydroxylase is phosphorylated on serine 40 by an associated protein kinase

Tyrosine hydroxylase, a key enzyme in the biosynthesis of catecholamines, was previously shown to be phosphorylated on four distinct serine residues in PC12 cell cultures, each one being specific for the kinase system involved (McTigue, M., Cremins, J., and Halegoua, S. (1985) J. Biol. Chem. 260, 9047-9056). A cAMP- and Ca2+-independent protein kinase was found to be associated with tyrosine hydroxylase purified from rat pheochromocytoma tumor. The use of this activity and the availability of a large amount of purified tyrosine hydroxylase allowed identification of the site phosphorylated by this kinase activity. A peptide of 1.5 kDa (about 12 residues long), carrying the phosphorylation site, was released from 32P-labeled tyrosine hydroxylase by limited proteolysis with trypsin. This peptide was isolated from trypsinized tyrosine hydroxylase by sequential gel filtration and ion exchange chromatographies. Analysis by thin layer chromatography of an acid hydrolysate of the peptide revealed that it contained phosphoserine. The sequence determination of the peptide showed that it corresponded to the residues 38-45 in the tyrosine hydroxylase primary structure (Arg-Gln-Ser(P)-Leu-Ile-Glu-Asp-Ala). Thus, the associated kinase phosphorylated Ser-40, one of the phosphorylation sites for the cAMP-dependent protein kinase also found in rat pheochromocytoma tumors. These results are compared to those recently appearing in a report by Campbell et al. (Campbell, D. G., Hardie, D. G., and Vulliet, P. R. (1986) J. Biol. Chem. 261, 10489-10492).     

Genetic variations in the SPP1 promoter affect gene expression and the level of osteopontin secretion into bovine milk

Osteopontin (OPN) is now recognized as an important cytokine and extracellular integrin‐binding protein at the crossroads of inflammation and homeostasis. In a previous study, we found that OPN gene (SPP1) polymorphisms are associated with milk performance traits and somatic cell score (SCS), a parameter used to estimate the genetic value of udder health in dairy cattle. In this study, we assessed whether the genetic variations had an impact on SPP1 promoter activity, immune response and the level of OPN secreted into milk. The influence of DNA polymorphisms on the promoter activity of SPP1 was confirmed in vitro. To measure the impact of the genetic variations on OPN secretion into milk, we measured OPN levels in both plasma and milk throughout lactation. Cows were grouped by the OPN haplotypes associated with a high (H2 × H3) or low (H1 × H4) SCS. For both H2 × H3 and H1 × H4, the OPN level in plasma remained low throughout lactation, although the concentration in the milk of H1 × H4 cows increased more in late lactation. Moreover, the macrophages of H1 × H4 cows expressed a lower SPP1 and proinflammatory IL6 in response to infection. Regarding the immune cell response, cows with the genetic potential to secrete higher OPN levels during late lactation had macrophages expressing fewer proinflammatory cytokines, a situation that might explain the genetic association with low somatic cells. Although OPN's favorable roles during late lactation remain to be elucidated, the tissue remodeling properties associated with OPN may be beneficial for reducing the incidence of infection during the transition period in lactating cows.     

Structural Maintenance of Chromosome (SMC) Proteins Link Microtubule Stability to Genome Integrity

Structural maintenance of chromosome (SMC) proteins are key organizers of chromosome architecture and are essential for genome integrity. They act by binding to chromatin and connecting distinct parts of chromosomes together. Interestingly, their potential role in providing connections between chromatin and the mitotic spindle has not been explored. Here, we show that yeast SMC proteins bind directly to microtubules and can provide a functional link between microtubules and DNA. We mapped the microtubule-binding region of Smc5 and generated a mutant with impaired microtubule binding activity. This mutant is viable in yeast but exhibited a cold-specific conditional lethality associated with mitotic arrest, aberrant spindle structures, and chromosome segregation defects. In an in vitro reconstitution assay, this Smc5 mutant also showed a compromised ability to protect microtubules from cold-induced depolymerization. Collectively, these findings demonstrate that SMC proteins can bind to and stabilize microtubules and that SMC-microtubule interactions are essential to establish a robust system to maintain genome integrity.     

Influence of the substituents of the carboxyl groups and of the rhamnose content on the solution properties and flexibility of pectins

Viscometric measurements were carried out on well-characterized apple, citrus, sugar-beet pectins in order to analyse the effect of the nature and the amount of substituents (methyl, amide, acetyl groups) and of the rhamnose content on the flexibility of the polymeric backbone. Through the dependence of the intrinsic viscosity with the ionic strength the flexibility parameter B was determined. B values between 0.072 and 0.017 indicate that pectins are relatively stiff molecules. However, an increase in flexibility is noticeable with the rise of the rhamnose content and of the amount of amide groups of the pectic acids. The flexibility is also sensitive to the degree of methylation.