Novel glycosylated VIP analogs: synthesis, biological activity, and metabolic stability.

Vasoactive intestinal peptide (VIP) is a prominent neuropeptide, exhibiting a wide spectrum of biological activities in mammals. However, the clinical applications of VIP are mainly hampered because of its rapid degradation in vivo. Peptide glycosylation, a procedure frequently used to increase peptide resistance to proteolytic degradation and consequently increase peptide metabolic stability, has not been performed yet on VIP. The presence of three N-glycosylation sites on VIP receptor type 1 (VPAC1) was previously demonstrated. Therefore, glycosylation of the VIP ligand could potentially increase its receptor affinity because of glyco-glyco interactions between the ligand and the receptor. In order to enhance VIP's metabolic stability and to increase its ligand-receptor binding/activation, eight glycosylated VIP derivatives were successfully synthesized by the solid-phase procedure. Each VIP analog was monoglycosylated by a monosaccharide addition to one amino-acid residue along the sequence. Glycosylation did not affect the alpha-helical structure shown by the native VIP in organic environment. Few glycosylated VIP analogs displayed highly potent VPAC1 receptor binding and cAMP-induced activation; only 4-6 fold lower in comparison to the native VIP. Furthermore, the peptide analog glycosylated on Thr11 ([11Glyc]VIP) showed a significantly enhanced stability toward trypsin enzymatic degradation in comparison to VIP. Analysis of the degradation products of [11Glyc]VIP showed that differently from VIP, incubation of the peptide [11Glyc]VIP with trypsin resulted in no cleavage at the Arg12-Leu13 peptide bond, suggesting that VIP glycosylation may lead to enhanced metabolic stability.     

Characterization of a new degradable polymer scaffold for regeneration of the dermis: In vitro and in vivo human studies

Full thickness skin wounds in humans heal with scars, but without regeneration of the dermis. A degradable poly(urethane urea) scaffold (PUUR), Artelon® is already used to reinforce soft tissues in orthopaedics, and for treatment of osteoarthritis of the hand, wrist and foot. In this paper we have done in vitro experiments followed by in vivo studies to find out whether the PUUR is biocompatible and usable as a template for dermal regeneration. Human dermal fibroblasts were cultured on discs of PUUR, with different macrostructures (fibrous and porous). They adhered to and migrated into the scaffolds, and produced collagen. The porous scaffold was judged more suitable for clinical applications and 4 mm Ø, 2 mm-thick discs of porous scaffold (12% w/w or 9% w/w polymer solution) were inserted intradermally in four healthy human volunteers. The implants were well tolerated and increasing ingrowth of fibroblasts was seen over time in all subjects. The fibroblasts stained immunohistochemically for procollagen and von Willebrand factor, indicating neocollagenesis and angiogenesis within the scaffolds. The PUUR scaffold may be a suitable material to use as a template for dermal regeneration.Key words: dermal regeneration, tissue engineering, polymer scaffold, wound healing, in vitro, in vivo, guided tissue regeneration, human, burns     

SIMPROT: Using an empirically determined indel distribution in simulations of protein evolution

Background  

General protein evolution models help determine the baseline expectations for the evolution of sequences, and they have been extensively useful in sequence analysis and for the computer simulation of artificial sequence data sets.     

Methyl jasmonate disrupts shoot formation in tobacco thin cell layers by over-inducing mitotic activity and cell expansion

The aim of the present study was to determine early cyto-histological events associated with the reduced number of shoots formed at the end of culture in tobacco (Nicotiana tabacum L.) thin cell layers treated with methyl jasmonate (MJ) [S. Biondi et al. (2001) J Exp Bot 52:1–12]. The results show that 0.1–10 M MJ strongly stimulated mitotic activity early in culture relative to untreated controls. Treatment with MJ also induced anomalous mitoses. Enhanced proliferative growth was confirmed by northern analysis and in situ hybridisation using cDNA probes of the G1/S phase-specific genes ubiquitin carboxyl-extension protein (ubi-CEP), topoisomerase 1 (top1) and ribonucleotide reductase (RNR). The formation of meristematic cell clusters on day 5 was also enhanced by 1 M MJ, but subsequent development of these cell clusters into meristemoids and shoot primordia was reduced by all MJ concentrations in a dose-dependent manner. Cell expansion was stimulated by MJ concentrations ranging from 0.001 to 10 M; expanded cells frequently occurred around and within meristemoids and shoot primordia, and displayed thickened and suberised cell walls; cell wall thickness increased with increasing MJ concentration. These cytological events caused alterations in the tunica and stem differentiation of the shoot dome. The apparently paradoxical role of MJ, which deregulates shoot formation through a stimulation of growth events, i.e., mitotic activity and cell expansion, is discussed.     

Presence of fetal DNA in maternal plasma decades after pregnancy

Cells of fetal origin and cell-free fetal DNA can be detected in the maternal circulation during pregnancy, and it has recently been shown that fetal cells can persist long after delivery. Given the various biological and clinical implications of this fact, we tested the hypothesis that cell-free fetal DNA can be present in maternal plasma decades after pregnancy. We extracted DNA from plasma samples and nucleated blood cells of 160 healthy women with male offspring at different time intervals after delivery (range 1-60 years). All of the samples were tested by means of a real-time quantitative PCR assay for a specific Y chromosome sequence (the SRY gene). Y chromosome-specific DNA was detected in 16 peripheral blood cell samples (10%) and 35 plasma samples (22%). The women with male sequences in the cell fraction had significantly greater total parity ( P=0.018). The proportion of women with detectable Y sequences in the plasma or cell samples was not related to the time since delivery. The fetal DNA concentrations in the genomic material extracted from plasma samples were significantly higher than those extracted from the Y-positive cell samples (149+/-140 vs 20+/-13 genome-equivalents/ml; P<0.001). There was no relationship between the concentration of fetal DNA and the time since delivery. Not only fetal cells, but also fragments of fetal DNA can be present in the maternal circulation indefinitely after pregnancy. This finding has practical implications for non-invasive prenatal diagnoses based on maternal blood, and may be considered for possible pathophysiological correlations.     

Conformational constraints of tyrosine in protein tyrosine kinase substrates: Information about preferred bioactive side-chain orientation

The side-chain orientation of a tyrosine residue located in a peptide, which is an excellent substrate of Syk tyrosine kinase (A. M. Brunati, A. Donella-Deana, M. Ruzzene, O. Marin, L. A. Pinna, FEBS Letters, 1995, Vol. 367, pp. 149-152), was fixed in the gauche (+) or gauche (-) conformation by using the 7-hydroxy-1,2,3,4-tetrahydro isoquinoline-3-carboxylic (Htc) structure. The tyrosine trans conformation was blocked by using an aminobenzazepine-type (Hba) structure. The proposed side-chain orientations were confirmed by the analysis of the (1)H-NMR parameters: chemical shifts, coupling constants, and nuclear Overhauser effects to the tyrosine constraints in the different analogs. This "rotamer scan" of the phosphorylatable residue allowed us to generate optimal substrates in terms of both phosphorylation efficiency and selectivity for Syk tyrosine kinase. In contrast, these conformationally restricted tyrosine analogs were not tolerated by the Src-related tyrosine kinases Lyn and c-Fgr.     

Lipid nutrition of Saccharomyces cerevisiae in winemaking

Biosynthesis of cell membrane lipids is a crucial metabolic pathway for the growth and viability of eucaryotic microorganisms. In Saccharomyces cerevisiae, unsaturated fatty acids and ergosterol synthesis needs molecular oxygen. Stuck and sluggish fermentations are related to this aspect of metabolism and constitute a major problem in the wine industry. Anaerobiosis, when lipids are not available in the growth medium, highly stresses cells. They release lipid biosynthesis metabolites and soon cease to multiply. This paper describes an investigation of the nutritional role of exogenous lipids from inactivated yeast cells (IYCs). Fermentations were carried out in a nitrogen-rich synthetic medium similar to grape juice with glucose and fructose as carbon sources, without lipid sources, and in anaerobiosis. The effect of the addition of IYC was assessed. Cell growth, cell lipid composition, glucose and fructose consumption, and acetic acid production were measured during fermentation. Addition of IYC boosted cell growth and sugar consumption, whereas acetic acid production decreased. Biomass yield was influenced by ergosterol availability and increased when IYCs were added. Fatty acid composition of yeast cells was changed by IYC addition.     

Plasma membrane oxidoreductase activity in cultured cells in relation to mitochondrial function and oxidative stress

Dichlorophenol indophenol (DCIP) reduction by intracellualr pyridine nucleotides was investigated in two different lines of cultured cells characterized by enhanced production of reacive oxygen species (ROS) with respect to suitable controls. The first line denominated XTC-UC1 was derived from a metastasis of an oxyphilic thyroid tumor characterized by mitochondrial hyperplasia and compared with a line (B-CPAP) derived from a papillary thyroid carcinoma with normal mitochondrial mass. The second line (170 MN) was a cybrid line derived from rho0 cells from an osteosarcoma line (143B) fused with platelets from a patient with a nucleotide 9957 mutation in mitochondrial DNA (encoding for cytochrome c oxidase subunit III) in comparison with the parent 143B line. The experimental lines had no major decreases of electron transfer activities with respect to the controls; both of them, however, exhibited an increased peroxide production. The XTC-UC1 cell line exhibited enhanced activity with respect to control of dicoumarol-sensitive DCIP reduction, identified with membrane bound DT-diaphorase, whereas dicoumarol insensitive DCIP reduction was not significantly changed. On the other hand the mtDNA mutated cybrids exhibited a strong increase of both dicoumarol sensitive and insensitive DCIP reduction. The results suggest that enhanced oxidative stress and not deficient respiratory activity per se is the stimulus triggering over-expression of plasma membrane oxidative enzymes.     

Design, synthesis and in vitro evaluation on HRPE cells of ascorbic and 6-bromoascorbic acid conjugates with neuroactive molecules

Preliminary investigations allowed us to anticipate that conjugation of nipecotic acid with L-ascorbate (AA) gave a prodrug endowed with anticonvulsant activity in mice. In view of these results, and in order to get deepen insight into the molecular aspects at the base of the transport mechanism, a second generation of compounds, based on 6-bromo-6-deoxy-L-ascorbic acid (BrAA) as the carrier molecule was designed and synthesized. Effects of the chirality of the transported drug was also investigated on R- and S-nipecotic acid. Interaction and uptake modalities were evaluated in our in vitro model based on human retinal pigment epithelium cells (HRPE), which expresses the membrane L-ascorbic acid (AA) SVCT2 transporters. A remarkable increase on SVCT2 affinity was found going from AA to BrAA conjugates, that is, 11 (Ki=1187+/-78 microM) versus 19 (Ki=193+/-14 microM) and 12 (Ki=39.8+/-3.2 microM) versus 20, (Ki=7.4+/-0.8 microM). Taken together, these data are in agreement with our initial hypothesis on the possibility to achieve better affinities by conjugation with AA analogs, and also consent to hypothesize the presence of accessory interactions that may improve transporters recognition.