Monitoring of native chemical ligation on solid substrate by surface plasmon resonance

During the last years native chemical ligation (NCL) gained in popularity as a method allowing the chemical synthesis of large peptides and entire proteins. NCL is particularly well-suited for chemoselective and nondenaturing attachment of biomolecules on solid substrates. In the present work, we show the feasibility of monitoring of peptide synthesis, NCL and its catalysis on silicon oxide modified gold surfaces by surface plasmon resonance (SPR). NCL of a model peptide-bradykinin thioester-was carried out and monitored with a custom-built SPR apparatus. Solid-phase produced bradykinin thioester was ligated to the surface in the presence of variable concentrations of 4-mercaptophenylacetic acid as transthioesterification catalyst. At catalyst concentration of 48 mM and above, the NCL reaction was maximal and identical to the reaction of the purified peptide-mercaptophenylacetic acid thioester. SPR curves indicate typical first-order kinetics with t(1/2) of 81 s for this aryl thioester, but of 104 min for the primary alkyl thioester.     

Effects of epidermal growth factor (EGF) on adult mouse small intestine in vivo and in organ culture

1. Exogenous administration of epidermal growth factor (EGF) has not modified the protein and DNA content, nor several brush border enzymes activities of duodenum, jejunum and ileum of intact and fasted adult mice. 2. Exogenous administration of EGF has not stimulated the DNA synthesis in the three regions of the small intestine of intact adult mice. 3. EGF has a stimulatory effect on DNA synthesis of fasted mice intestine 12 hr after injection. 4. In organ culture, EGF has not altered at any concentration (10, 50, 100, 200, 800 ng/ml), the same parameters in duodenal and jejunal explants taken from animals fasted 24 hr before being killed. 5. These last results suggest that the increase of DNA synthesis observed in vivo was not a direct effect of EGF administration. 6. Finally, the EGF content of serum af adult male mice was measured in fed and fasted mice and in the organ culture media.     

Human exposure to mycotoxins in Egypt

This investigation examined the exposure of Egyptian infants to Aflatoxin M₁ (AfM₁) and of lactating mothers to Aflatoxin B₁, using AfM₁ in human milk as a biomarker for exposure to AfB₁. The presence of ochratoxin A (OA) in human milk was also investigated to determine the levels of infants exposure to OA from human milk. The results indicated that AfM₁ was found in 66 (55 %) of 120 human milk samples with a mean of 0.3 ± 0.53 ng/mL (range 0.02 to 2.09 ng/mL). OA was found in 43 (35.8 %) of 120 human milk samples with a mean of 21.1 ± 13.7 ng/mL (range 5.07 to 45.01 ng/mL), which will cause a daily intake of OA from human milk exceeding the suggested tolerable dose of 5 ng/kg_-1 of OA body weight. On the other side AfM₁ was found in 25 % of blood samples (5 out of 20 samples), at a mean of 1.18 ng/mL, but it was detected only in one urine sample (1 out of 20 samples). OA was detected only in 2 out of 13 blood samples (15.4 %) with an average 3.67 ng/mL. Whereas OA was not detected in all analyzed urine samples.     

Rhythms of locomotion expressed by Limulus polyphemus, the American horseshoe crab: I. Synchronization by artificial tides

Limulus polyphemus, the American horseshoe crab, has an endogenous clock that drives circatidal rhythms of locomotor activity. In this study, we examined the ability of artificial tides to entrain the locomotor rhythms of Limulus in the laboratory. In experiments one and two, the activity of 16 individuals of L. polyphemus was monitored with activity boxes and "running wheels." When the crabs were exposed to artificial tides created by changes in water depth, circatidal rhythms were observed in animals exposed to 12.4-h "tidal" cycles of either water depth changes (8 of 8 animals) or inundation (7 of 8 animals). In experiment three, an additional 8 animals were exposed to water depth changes under cyclic conditions of light and dark and then monitored for 10 days with no imposed artificial tides. Most animals (5) clearly synchronized their activity to the imposed artificial tidal cycles, and 3 of these animals showed clear evidence of entrainment after the artificial tides were terminated. Overall, these results demonstrate that the endogenous tidal clock that influences locomotion in Limulus can be entrained by imposed artificial tides. In the laboratory, these tidal cues override the influence of light/dark cycles. In their natural habitat, where both tidal and photoperiod inputs are typically always present, their activity rhythms are likely to be much more complex.     

Modulation of exon skipping by high-affinity hnRNP A1-binding sites and by intron elements that repress splice site utilization

The RNA-binding protein hnRNP A1 is a splicing regulator produced by exclusion of alternative exon 7B from the A1 pre-mRNA. Each intron flanking exon 7B contains a high-affinity A1-binding site. The A1-binding elements promote exon skipping in vivo, activate distal 5' splice site selection in vitro and improve the responsiveness of pre-mRNAs to increases in the concentration of A1. Whereas the glycine-rich C-terminal domain of A1 is not required for binding, it is essential to activate the distal 5' splice site. Because A1 complexes can interact simultaneously with two A1-binding sites, we propose that an interaction between bound A1 proteins facilitates the pairing of distant splice sites. Based on the distribution of putative A1-binding sites in various pre-mRNAs, an A1-mediated change in pre-mRNA conformation may help define the borders of mammalian introns. We also identify an intron element which represses the 3' splice site of exon 7B. The activity of this element is mediated by a factor distinct from A1. Our results suggest that exon 7B skipping results from the concerted action of several intron elements that modulate splice site recognition and pairing.     

Functional impairment of MSC induced by transient warming events: Correlation with loss of adhesion and altered cell size

Background

We recently showed that transient warming effects decreased the functional and adhesion properties of mesenchymal stromal cells (MSC) while post-thaw viability remained high. In an attempt to better predict functional impairment of cryopreserved MSC, we further analysed the correlation between viability, immunosuppressive activity and adhesion of cells exposed or not to warming events.

Evolutionary origin of human and primate malarias: evidence from the circumsporozoite protein gene

We have analyzed the conserved regions of the gene coding for the circumsporozoite protein (CSP) in 12 species of Plasmodium, the malaria parasite. The closest evolutionary relative of P. falciparum, the agent of malignant human malaria, is P. reichenowi, a chimpanzee parasite. This is consistent with the hypothesis that P. falciparum is an ancient human parasite, associated with humans since the divergence of the hominids from their closest hominoid relatives. Three other human Plasmodium species are each genetically indistinguishable from species parasitic to nonhuman primates; that is, for the DNA sequences included in our analysis, the differences between species are not greater than the differences between strains of the human species. The human P. malariae is indistinguishable from P. brasilianum, and P. vivax is indistinguishable from P. simium; P. brasilianum and P. simium are parasitic to New World monkeys. The human P. vivax-like is indistinguishable from P. simiovale, a parasite of Old World macaques. We conjecture that P. malariae, P. vivax, and P. vivax-like are evolutionarily recent human parasites, the first two at least acquired only within the last several thousand years, and perhaps within the last few hundred years, after the expansion of human populations in South America following the European colonizations. We estimate the rate of evolution of the conserved regions of the CSP gene as 2.46 x 10(-9) per site per year. The divergence between the P. falciparum and P. reichenowi lineages is accordingly dated 8.9 Myr ago. The divergence between the three lineages leading to the human parasites is very ancient, about 100 Myr old between P. malariae and P. vivax (and P. vivax-like) and about 165 Myr old between P. falciparum and the other two.