(+)-4-phosphonophenylglycine (PPG) a new group III selective metabotropic glutamate receptor agonist

A new synthesis of (R,S)-PPG (4-phosphonophenylglycine) and the separation of the protected enantiomers leading after deprotection to (+)- and (-)-PPG are described. Pharmacological characterization at the group III metabotropic glutamate receptors hmGluR4a and hmGluR7b revealed (+)-PPG as the active enantiomer.     

Effect of microwave irradiation on phosphoramidite couplings on controlled pore glass

The chain extension step in the synthesis of DNA oligomers on controlled pore glass was shown to be higher yielding when the reaction mixture is irradiated with microwaves. Both a commercial thymidine 3'-phosphoramidite building block and a 3'-phosphoramidite of protected 1'-aminomethylthymidine were coupled using dilute solutions that give only partial conversion. In either case, higher coupling yields were observed when microwaves were used. The results of our exploratory experiments suggest that microwave-assisted DNA syntheses might require fewer equivalents of phosphoramidites and/or shorter coupling times than those performed at room temperature.     

Proteomic analysis identifies novel proteins of the Maurer's clefts, a secretory compartment delivering Plasmodium falciparum proteins to the surface of its host cell

A novel method was validated for the efficient distinction between malaria parasite-derived and host cell proteins in mass spectrometry analyses. This method was applied to a ghost fraction from Plasmodium falciparum-infected erythrocytes containing the red blood cell plasma membrane, the erythrocyte submembrane skeleton, and the Maurer's clefts, a Golgi-like apparatus linked to and addressing parasite proteins to the host cell surface. This method allowed the identification of 78 parasite proteins. Among these we identified seven novel proteins of the Maurer's clefts based on immunofluorescence studies and proteinase K digestion assays. The products of six contiguous genes located on chromosome 5 were identified, and the location within the Maurer's clefts was established for two of them. This suggests a clustering of genes encoding Maurer's cleft proteins. Our study sheds new light on the biological function of the Maurer's clefts, which are central to the pathogenesis and to the intraerythrocytic development of P. falciparum.     

Increasing the frequency of co-mingling piglets during the lactation period alters the development of social behavior before and after weaning

The purpose of this study was to determine how increasing the frequency of co-mingling affected piglets' behavior development before and after weaning. Co-mingling once (CM1), piglets interacted with 1 unfamiliar litter Days 10-18 after birth; co-mingling twice (CM2), piglets interacted with 1 unfamiliar litter Days 10-14; and with another, Days 14-18. Control (CM0) piglets did not interact with unfamiliar litters before Day 18 (n = 16 litters per treatment). The study weighed piglets and recorded ear-injury scores throughout the experiment; however, there were no treatment differences. The CM2 piglets spent less time engaged in aggressive interactions (p < .05) than did CM0 piglets following mixing at weaning. During the social challenge, CM2 piglets spent more time in proximity to one another, had shorter latencies to first aggressive interaction, and spent less time fighting than did CM0 piglets (p < .05). During the social recognition test, CM2 piglets recognized the stimulus piglet at a faster rate than did CM0 piglets (p < .05). Overall, CM2 resulted in changes to some social behaviors compared with CM0 but not with CM1.     

Predicting substrate specificity of adenylation domains of nonribosomal peptide synthetases and other protein properties by latent semantic indexing

Successful genome mining is dependent on accurate prediction of protein function from sequence. This often involves dividing protein families into functional subtypes (e.g., with different substrates). In many cases, there are only a small number of known functional subtypes, but in the case of the adenylation domains of nonribosomal peptide synthetases (NRPS), there are >500 known substrates. Latent semantic indexing (LSI) was originally developed for text processing but has also been used to assign proteins to families. Proteins are treated as ‘‘documents’’ and it is necessary to encode properties of the amino acid sequence as ‘‘terms’’ in order to construct a term-document matrix, which counts the terms in each document. This matrix is then processed to produce a document-concept matrix, where each protein is represented as a row vector. A standard measure of the closeness of vectors to each other (cosines of the angle between them) provides a measure of protein similarity. Previous work encoded proteins as oligopeptide terms, i.e. counted oligopeptides, but used no information regarding location of oligopeptides in the proteins. A novel tokenization method was developed to analyze information from multiple alignments. LSI successfully distinguished between two functional subtypes in five well-characterized families. Visualization of different ‘‘concept’’ dimensions allows exploration of the structure of protein families. LSI was also used to predict the amino acid substrate of adenylation domains of NRPS. Better results were obtained when selected residues from multiple alignments were used rather than the total sequence of the adenylation domains. Using ten residues from the substrate binding pocket performed better than using 34 residues within 8 Å of the active site. Prediction efficiency was somewhat better than that of the best published method using a support vector machine.     

Cell stiffening in response to external stress is correlated to actin recruitment

We designed a micromanipulation device that allows the local application of a constant force on living cells, and the measurement of their stiffness. The force is applied through an Arg-Gly-Asp-coated bead adhering on the cell and trapped in optical tweezers controlled by a feedback loop. Epifluorescence observations of green fluorescent protein-actin in the cells are made during force application. We observe a stiffening of cells submitted to a constant force within a few minutes, coupled to actin recruitment both at the bead-cell contact and up to several micrometers from the stress application zone. Moreover, kinetics of stiffening and actin recruitment exhibit a strong correlation. This work presents the first quantification of the dynamics of cell mechanical reinforcement under stress, which is a novel insight into the elucidation of the more general phenomenon of cell adaptation to stress.     

Synthesis and monitored selection of nucleotide surrogates for binding T:A base pairs in homopurine-homopyrimidine DNA triple helices

A total of 16 oligodeoxyribonucleotides of general sequence 5′-TCTTCTZTCTTTCT-3′, where Z denotes an N-acyl-N-(2-hydroxyethyl)glycine residue, were prepared via solid phase synthesis. The ability of these oligonucleotides to form triplexes with the duplex 5′-AGAAGATAGAAAGA-HEG-TCTTTCTATCTTCT-3′, where HEG is a hexaethylene glycol linker, was tested. In these triplexes, an ‘interrupting’ T:A base pair faces the Z residue in the third strand. Among the acyl moieties of Z tested, an anthraquinone carboxylic acid residue linked via a glycinyl group gave the most stable triplex, whose UV melting point was 8.4°C higher than that of the triplex with 5′-TCTTCTGTCTTTCT-3′ as the third strand. The results from exploratory nuclease selection experiments suggest that a combinatorial search for strands capable of recognizing mixed sequences by triple helix formation is feasible.     

Hepatocyte ploidy in normal young rat

The aim of the present study was to examine the relation between hepatocyte size and ploidy in Sprague-Dawley rat liver. Therefore, subpopulations of hepatocytes of various sizes were separated from the isolated crude hepatocyte population either mechanically or by using centrifugal elutriation. Hepatocyte size was determined on scanning electron microscopy photographs. Ploidy of hepatocytes was assessed by flow cytometry. The crude hepatocyte population was very heterogeneous in sizes, with diameters ranging from 8 to 39 microm. Hepatocyte ultrastructure was well preserved as demonstrated by transmission electron microscopy. The distribution of hepatocytes within the ploidy classes was the following: 19.6+/-3.6% diploid, 56.2+/-3.2% tetraploid and 3.4+/-0.6% octoploid mononucleated cells. Thus approximately 79% of hepatocytes appeared mononucleated. The binucleated hepatocytes (21%) had two diploid nuclei (18.7+/-2.9%) or two tetraploid nuclei (2.1+/-0.6%). A similar distribution of hepatocytes into ploidy classes was obtained in subpopulations of hepatocytes of various sizes. Our findings suggest that distribution into ploidy classes is not strictly correlated with hepatocyte size. In accordance with previous observations, our results on hepatocyte ploidy from periportal or perivenous origin using digitonin perfusion, is in favour of the existence of ploidy zonation within the rat hepatic lobule.