New diketopiperazines as vectors for peptide protection and brain delivery: Synthesis and biological evaluation.

New strategies allowing the transfer of molecules, especially peptides, through the blood-brain barriers are a major pharmacological challenge for the treatment of brain diseases. The present study aims at evaluating in vivo the cerebral bioavailability of carrier systems, based on small and functionalizable 2,5-diketopiperazine (DKP) motifs. We studied 2 different cyclo(Lys-Lys) DKP scaffolds alone and a cyclo(Lys-Gly) DKP carrier bearing as peptide model, the tau protein hexapeptide VQIVYK sequence. The different carrier systems were synthesized and radiolabeled using one of the free domains. The stability, biodistribution, and ability to cross blood-brain barrier were investigated in vivo in mice for 99m Tc-DKP scaffolds, 99m Tc-HVQIVYK peptide alone, and 99m Tc-DKP-VQIVYK. 125 I-labelled bovine serum albumin was used as negative control for brain uptake. Both radiolabeled DKPs scaffolds and 99m Tc-DKP-VQIVYK showed a high stability, while peptide 99m Tc-HVQIVYK alone was quickly degraded in vivo. The presence of 99m Tc-DKPs scaffolds and 99m Tc-DKP-VQIVYK was observed in the ventricular and subarachnoid spaces and to a lower extent in the brain parenchyma up to 45 minutes post-injection in mice. This work highlights the potentiality of DKP scaffolds as vectors to transport peptides into the brain by limiting proteolysis and favoring cerebral bioavailability.

'Clickable' 2,5-diketopiperazines as scaffolds for ligation of biomolecules: their use in Aß inhibitor assembly.

The synthesis of 1,3,6-trisubstituted-2,5-diketopiperazine scaffolds bearing up to three 'clickable' sites for further oxime bond or alkyne-azide cycloaddition ligations is described. The orthogonally Boc/Alloc protected DKP precursors prepared from L-lysine residues and an aminohexyl arm are efficiently prepared on a gram scale by sequentially using Fukuyama-Mitsunobu alkylation, dipeptide coupling and diketopiperazine ring formation as key steps. These scaffolds, with their glyoxylyl, aminooxy, alkynyl or azido functions, are "ready-to-use" platforms for biomolecular assembly. Their potentiality in this field was proved through the chemoselective ligation of Aß-binding motifs, the KLVFFA peptide and the curcumin molecule. The inhibitory effect of these conjugates on Aß amyloid fibril formation is reported using thioflavin T fluorescence assays and AFM observation.

Front-face fluorescence spectroscopy as a tool to classify seven bovine muscles according to their chemical and rheological characteristics.

Potential of front-face fluorescence spectroscopy was evaluated to classify muscles according to their chemical and rheological characteristics. Seven bovine muscles (Semitendinosus, Semimembranosus, Tensor fasciae latae, Rectus abdominis, Longissimus thoracis et lumborum, Triceps branchii and Infraspinatus) were taken from 14 animals of the Charolais breed. Chemical characteristics and rheological properties of the meat were determined including dry matter, fat, collagen, protein, peak load, energy required to rupture and cooking loss. Emission spectra in the 305-400nm, 340-540nm and 410-700nm ranges were recorded using front-face fluorescence spectroscopy by fixing the excitation wavelengths at 290, 322 and 382nm, respectively. Analysis of variance (ANOVA) applied on chemical and rheological parameters showed that these muscles were significantly different (P<0.01) from each other. Chemical and rheological data were divided into low, medium and high range groups for each variable. The results of PLSDA showed that 305-400nm spectra were responsible for 67% (calibration), 53% (validation), 96% (calibration) and 55% (validation) of good classification for protein and cooking loss, respectively, while 340-540nm spectra allowed 75% of good classification (validation samples) for fat content.

Sphingosine kinase 1 overexpression induces MFN2 fragmentation and alters mitochondrial matrix Ca2+ handling in HeLa cells.

Sphingosine kinase 1 (SK1) converts sphingosine to the bioactive lipid sphingosine 1-phosphate (S1P). S1P binds to G-protein-coupled receptors (S1PR1-5) to regulate cellular events, including Ca2+ signaling. The SK1/S1P axis and Ca2+ signaling both play important roles in health and disease. In this respect, Ca2+ microdomains at the mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) are of importance in oncogenesis. Mitofusin 2 (MFN2) modulates ER-mitochondria contacts, and dysregulation of MFN2 is associated with malignancies. We show that overexpression of SK1 augments agonist-induced Ca2+ release from the ER resulting in increased mitochondrial matrix Ca2+. Also, overexpression of SK1 induces MFN2 fragmentation, likely through increased calpain activity. Further, expressing putative calpain-cleaved MFN2 N- and C-terminal fragments increases mitochondrial matrix Ca2+ during agonist stimulation, mimicking the SK1 overexpression in cells. Moreover, SK1 overexpression enhances cellular respiration and cell migration. Thus, SK1 regulates MFN2 fragmentation resulting in increased mitochondrial Ca2+ and downstream cellular effects.

Human percutaneous absorption of a direct hair dye comparing in vitro and in vivo results: implications for safety assessment and animal testing.

Although in vitro skin absorption studies often detect small residues of applied test material in the epidermis/dermis, it is uncertain whether the residue is within the living skin. We studied the dermal absorption of a hair dye hydroxyanthraquinone-aminopropyl methyl morpholinium methosulphate (HAM) in human skin in vivo and in vitro. In vivo, skin (back and scalp) received 0.5% HAM in a commercial formulation at 20microg/cm2 After 0.5 or 48h, skin was tape stripped, followed by cyanoacrylate biopsies (CAB). Sebum from scalp sites was collected for 48h. In vitro, skin was treated with 20mg/cm2 dye for 0.5h, penetration determined after 24h. In vivo, at 0.5h, total recovery (back) was 0.67microg/cm2 (tape strips+CAB). Fluorescence microscopy showed HAM in the hair follicle openings (HFO). At 0.5h, scalp tape strips contained 1.80microg/cm2, HFO 0.82microg/cm2. At 48h, HFO contained 0.21microg/cm2, sebum 0.80microg/cm2. In vivo, skin residues were in the non-living skin and eliminated via desquamation and sebum secretion. In vitro, the SC contained 1.50microg/cm2, epidermis/dermis 0.86microg/cm2, receptor fluid<0.04microg/cm2, a total of 0.90microg/cm2 was considered to be bioavailable. In vitro epidermis/dermis residues were nearly identical to those located in non-living skin in vivo. In conclusion, in vitro percutaneous penetration studies may produce seemingly bioavailable material , which raises the need for a Threshold of Skin Absorption (TSA) addressing a negligible dermal absorption in order to avoid unnecessary in vivo toxicity studies on substances that produce no significant human systemic exposure.

Nanotechnology, cosmetics and the skin: is there a health risk?

Cosmetic formulations may contain nano-emulsions and microscopic vesicles consisting of traditional cosmetic materials, although it is uncertain whether they should be qualified as actual nanomaterials. Vesicle materials do not penetrate into living human skin. Vesicle formulations may enhance or reduce skin absorption of ingredients, albeit at a limited scale. Sunscreens contain TiO2 or ZnO nanoparticles (NP), which are efficient UV filters. A number of studies suggest that insoluble NP do not penetrate into or through human skin. The results of in vivo toxicity tests showed that TiO2 and ZnO NP are non-toxic. In vitro and in vivo cytotoxicity, genotoxicity, photogenotoxicity, acute toxicity, sensitisation and ecotoxicology studies on TiO2 NP found no difference in the safety profile of micro- or nano-sized materials, all of which were non-toxic. Although some in vitro investigations on TiO2 particles reported cell uptake, oxidative cell damage or genotoxicity, these results may be secondary to phagocytosis of cells exposed to excessive particle concentrations. Studies on wear debris nano- and microparticles support the traditional view that toxicity of small particles is related to their chemistry, rather than their particle size. There is little evidence supporting a general rule that adverse effects of particles on the skin or other tissues increase with smaller particle size, or produce novel toxicities relative to those of larger particles. Overall, the current evidence suggests that nano-sized cosmetic or sunscreen ingredients pose no potential risk to human health, whereas their use in sunscreens has large benefits, such as the protection of human skin against skin cancer.

Mitochondrial group II introns, cytochrome c oxidase, and senescence in Podospora anserina.

Podospora anserina is a filamentous fungus with a limited life span. It expresses a degenerative syndrome called senescence, which is always associated with the accumulation of circular molecules (senDNAs) containing specific regions of the mitochondrial chromosome. A mobile group II intron (alpha) has been thought to play a prominent role in this syndrome. Intron alpha is the first intron of the cytochrome c oxidase subunit I gene (COX1). Mitochondrial mutants that escape the senescence process are missing this intron, as well as the first exon of the COX1 gene. We describe here the first mutant of P. anserina that has the alpha sequence precisely deleted and whose cytochrome c oxidase activity is identical to that of wild-type cells. The integration site of the intron is slightly modified, and this change prevents efficient homing of intron alpha. We show here that this mutant displays a senescence syndrome similar to that of the wild type and that its life span is increased about twofold. The introduction of a related group II intron into the mitochondrial genome of the mutant does not restore the wild-type life span. These data clearly demonstrate that intron alpha is not the specific senescence factor but rather an accelerator or amplifier of the senescence process. They emphasize the role that intron alpha plays in the instability of the mitochondrial chromosome and the link between this instability and longevity. Our results strongly support the idea that in Podospora, "immortality" can be acquired not by the absence of intron alpha but rather by the lack of active cytochrome c oxidase.

[Patient's experience of topical anesthesia by lidocaine vaginal gel for oocyte retrieval]. / Anesthésie topique par gel de lidocaïne intravaginal pour ponction ovocytaire : retour d'expérience.

A recent adverse effect of a paracervical block (cardiac arrest) occurred during an oocyte retrieval (OR), forcing us to reconsider our pain management during OR. Since then, we decided to use intravaginal lidocaine gel as analgesia during OR. OBJECTIVES: To evaluate the pain during OR after intravaginal lidocaine gel analgesia and to evaluate the motivations of women choosing this technique. METHODS: A monocentric observational study was performed on 200 patients. Pain was measured using a numeric pain scale during and after oocyte retrieval. The tolerance of the procedure was evaluated through a patient questionnaire. RESULTS: Median maximal pain was 5±2.3 (0-10) per-retrieval and 3±2.2 (0-10) post-retrieval. The procedure was considered bearable by 85.5% of the patients and 81.5% of them would choose this method in case of new oocyte retrieval. No adverse effect occurred during the study. CONCLUSION: The use of intravaginal lidocaine gel seems an acceptable analgesia alternative during oocyte retrieval.

Binding of retinoids and beta-carotene to beta-lactoglobulin. Influence of protein modifications.

The binding of retinol, retinyl acetate, retinoic acid and beta-carotene to native, esterified and alkylated beta-lactoglobulin was followed by quenching of tryptophan fluorescence. Three studied retinoids bind to native or modified beta-lactoglobulin in 1:1 molar ratios, with apparent dissociation constants in the range of 10(-8) M. The maximum tryptophan fluorescence quenching of unmodified beta-lactoglobulin by beta-carotene is observed at the ligand/protein ratio of 1:2. Esterification and alkylation of beta-lactoglobulin shift the ratio of beta-carotene/protein to 1:1. In all the cases, except for retinoic acid binding to N-ethyllysyl-BLG, the performed chemical modifications of beta-lactoglobulin enhance protein binding affinity. Measured apparent dissociation constants of beta-carotene complexes with native and modified beta-lactoglobulin are an order of magnitude lower from binding constants of other studied retinoids.

Delineation of chicken cathepsin L secondary structure; relationship between pH dependence activity and helix content.

The secondary structure of the recently sequenced chicken liver cathepsin L (EC 3.4.22.15) has been studied both by circular dichroism and a predictive method. The structural data provided by these approaches allow us to underline the extent of the structural similarities between cathepsin L and papain, one of the best known proteins in the cysteine proteinase family. The predictive method of Garnier et al. (J. Mol. Biol. 120 (1978) 97-120) is used to locate alpha-helix and beta-sheet segments in the cathepsin L sequence. An optimization of decision constants has been performed, using circular dichroism data, to improve good predictions. The combination of these approaches lead us to suggest that the location of ordered structures observed in papain is maintained in cathepsin L, but with an additional alpha-helix in the middle region (residues 85-108) of cathepsin L. Furthermore, we show that cathepsin L inactivation at neutral pH is correlated to the lost of alpha-helix content (40% at pH 5.8 and 17% at pH 7.0) in this protein. It appears that such an effect can be related to the change in the ionization state of histidine side-chains which are shown to be mainly located in the predicted alpha-helix regions.

beta-Lactoglobulin binding properties during its folding changes studied by fluorescence spectroscopy.

The milk protein, beta-lactoglobulin (BLG) exhibits structural and binding properties which vary widely, depending on the medium. These properties of BLG are reflected in fluorescence intensities, steady-state anisotropies and phase lifetimes of BLG tryptophan residues and of retinol and diphenyl hexatriene (DPH) bound to BLG, as functions of pH, ethanol concentration and protein modifications (22% ethylated, 90% methylated and 85% acetylated BLGs). Tryptophan quenching experiments show that retinol and DPH bind to BLG in 1:1 molar ratios with apparent dissociation constants around 10(-7) - 10(-8) M. The strength of retinol binding is pH-dependent in the range 3-8, whereas that of DPH binding is not. Two different binding sites for these two ligands coexist on the protein. Modified BLGs exhibit higher affinities for DPH than the unmodified protein. At all pH values investigated, the fluorescence emission at 480 nm of retinol/BLG mixtures and retinol, DPH and tryptophan anisotropies and lifetimes change dramatically with midpoint at 27% ethanol for the first parameter and 35% for the others, suggesting simultaneous beta-strand to alpha-helix transition and the dissociation of BLG complexes at 35% ethanol. An intermediate state, possibly 'molten globular', occurs around 20% ethanol, as deduced from anisotropy and lifetime measurements.

High-pressure effects on beta-lactoglobulin interactions with ligands studied by fluorescence.

The effects of pressure (0.1 MPa to 400 MPa) on intrinsic fluorescence of beta-lactoglobulin and on its binding of retinol and cis-parinaric acid have been studied at neutral and acid pHs. In neutral pH, fluorescence emission spectra of beta-lactoglobulin tryptophanes are characterized by an irreversible 14 nm red-shift indicating pressure-induced folding changes. The intensity of the fluorescence of retinol in beta-lactoglobulin-retinol complex is enhanced by a pressure increase up to 150 MPa. It decreases at higher pressures and disappears altogether at 300 MPa. beta-Lactoglobulin-retinol complex does not reassociate after decompression at neutral pH. At acid pH condition, the fluorescence quenching by pressure of beta-lactoglobulin tryptophans is coupled with a 2 nm spectral shift and is fully reversible demonstrating almost complete restoration of globulin folding. The evolution of retinol fluorescence in beta-lactoglobulin-retinol complex is also entirely reversible between 0.1 MPa and 400 MPa and the complex never dissociates in the studied pressure range. beta-lactoglobulin-cis-parinaric acid complexes at neutral and acid pH values dissociate irreversibly at 200 MPa and 350 MPa, respectively.

Influence of pH on the structural changes of beta-lactoglobulin studied by tryptic hydrolysis.

The attempt to use trypsin in order to monitor pH (7.5-9.0) induced beta-lactoglobulin conformation changes has revealed differences in the cleavage of specific sites. The tryptic cleavage of two dibasic X-Lys-Lys-Y sites (Lys 69, 70 and 100, 101) shows slighter predominance of symmetrical cut at pH 7.5 and 8.0. Mostly asymmetrical cleavage yielding two C-terminal lysines can be observed at pH 8.5 and 9.0. Atypical cleavage of the Tyr-20-Ser-21 site, which at pH 9.0 is relatively negligible, increases substantially in pH 7.5-8.5. This implies that Tyr-20 probably is the tyrosine reported to be exposed on the surface of the protein during transformation of beta-lactoglobulin molecule occurring in the studied pH range (Tanford et al. (1959) J. Am. Chem. Soc. 81, 4032-4036).

Human immunodeficiency virus type-1 reverse transcriptase copies very short templates: kinetic and crosslinking analysis.

We describe in this article some properties concerning the cDNA elongation activity of human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT). The kinetic parameters of the polymerization reaction catalyzed by HIV-1 RT, using short templates, were studied. Values of Km and Vmax were measured as a function of the oligoadenylate template length: the logarithm of Km increased linearly, with an incremental factor of 2.2, when the template length differs by one nucleotide. Using short templates, olig(A)n (n = 7-14) and primers shorter or longer than the template, HIV-1 reverse transcriptase was able to synthesize polymer products longer than 200 nucleotides. We showed that an oligonucleotide as short as (pA)3 was long enough to serve as template for cDNA synthesis by RT. In the binding of RT to template of different lengths (5 to 14 nucleotides long), two constants were determined differing in each case by a factor of about 10. The three recombinant forms of HIV-1 RT (p66/p51, p66/p66 and p51/p51) were crosslinked to a short template, (pA)14, in the presence of cis-aquahydroxydiamminoplatinum. The efficiency of crosslink of [32P](pA)14 template with each of the subunits of RT correlated well with the affinity of this template to the different forms of RT. In the case of p66/p51, the crosslink occurred mainly with the p66 subunit. These results confirm the important catalytic role of the p66 subunit in the heterodimeric human retroviral polymerase.

An aspartic acid residue in TPR-1, a specific region of protein-priming DNA polymerases, is required for the functional interaction with primer terminal protein.

A multiple sequence alignment of eukaryotic-type DNA polymerases led to the identification of two regions of amino acid residues that are only present in the group of DNA polymerases that make use of terminal proteins. (TPs) as primers to initiate DNA replication of linear genomes. These amino acid regions (named terminal region (TPR protein-1 and TPR-2) are inserted between the generally conserved motifs Dx(2)SLYP and Kx(3)NSxYG (TPR-1) and motifs Kx(3)NSxYG and YxDTDS (TPR-2) of the eukaryotic-type family of DNA polymerases. We carried out site-directed mutagenesis in two of the most conserved residues of phi29 DNA polymerase TPR-1 to study the possible role of this specific region. Two mutant DNA polymerases, in conserved residues AsP332 and Leu342, were purified and subjected to a detailed biochemical analysis of their enzymatic activities. Both mutant DNA polymerases were essentially normal when assayed for synthetic activities in DNA-primed reactions. However, mutant D332Y was drastically affected in phi29 TP-DNA replication as a consequence of a large reduction in the catalytic efficiency of the protein-primed reactions. The molecular basis of this defect is a non-functional interaction with TP that strongly reduces the activity of the DNA polymerase/TP heterodimer.

Cross-linking localization of a HIV-1 reverse transcriptase peptide involved in the binding of primer tRNALys3.

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) initiates the synthesis of DNA from the 3' end of its specific primer, tRNALys3. The regions of tRNALys3 in close contact with RT are well known, while a precise knowledge of the RT regions interacting with tRNALys3 is not yet available. To address this question we cross-linked the heterodimeric p66/p51 RT to tRNALys3 using cis-aquahydroxydiammino-platinum. Ribonucleoprotein complexes of molecular masses higher than the p66 subunit were obtained. After RNase A digestion of the RT-tRNA complex, a labeled oligoribonucleotide (ORN) was mainly found associated to the p66 subunit. This labeled p66-ORN complex was then proteolyzed with Staphylococcus aureus V8 protease. A highly purified radioactive peptide was obtained after two chromatographic purification steps. Its N-terminal sequence corresponded with amino acid residues 241VQPI244. Using the crystallographic structure of HIV-1 RT, this peptide was localized at the beta14-sheet end, near to the hairpin formed by beta12 and beta13-sheets ("primer grip") and the alphaH-helix. The so called "VQPI peptide" is in the border of the thumb and the palm subdomains of the p66 subunit. This study palliates the absence of a three- dimensional structure of the RT-tRNA complex and led to a peptide in interaction with tRNALys3 present in all HIV-1 RT isolates.

The effect of raw milk microbial flora on the sensory characteristics of Salers-type cheeses.

The sensory characteristics of Salers Protected Denomination of Origin raw-milk cheeses are linked to the biochemical composition of the raw material (milk) and to the resultant microbial community. To evaluate the influence of the microbial community on sensory characteristics, Salers-type cheeses were manufactured with the same pasteurized milk, reinoculated with 3 different microbial communities from 3 different filtrates from microfiltered milks. Each cheese was subjected to microbial counts (on selective media), biochemical tests, and volatile and sensory component analyses at different times of ripening. Adding different microbial communities to specimens of the same (biochemically identical) pasteurized milk lead to different sensory characteristics of the cheeses. Cheeses with fresh cream, hazelnut, and caramel attributes were opposed to those with fermented cream, chemical, and garlic flavors. The aromatic compounds identified (esters, acids, alcohols, and aldehydes) in these cheeses were quite similar. Nevertheless, one milk was distinguished by a higher content of acetoin, and lower 2-butanone and 3-methylpentanone concentrations. Over the production period of 1 mo, the different cheeses were characterized by the same balance of the microbial population assessed by microbial counts on different media. This was associated with the stability of some sensory attributes describing these cheeses. Nevertheless, there was no linear correlation between microbial flora data and sensory characteristics as measured in this study.

Beta-lactoglobulin binds retinol and protoporphyrin IX at two different binding sites.

Measurement of tryptophan fluorescence quenching and the excitation energy transfer from tryptophanyl residues to the bound ligand indicates that beta-lactoglobulin binds tightly to hemin and protoporphyrin IX in a ligand-to-protein stoichiometric ratio. The apparent dissociation constants of hemin-beta-lactoglobulin and protoporphyrin IX-beta-lactoglobulin complexes are 2.5 x 10(-7) M and 4 x 10(-7) M, respectively. The addition of beta-lactoglobulin (final concentration = 10 microM, phosphate buffer 50 mM, pH 7.1) to the solution containing retinol and protoporphyrin IX triggers an energy transfer between beta-lactoglobulin tryptophan and protoporphyrin IX as well as between retinol and protoporphyrin IX. The efficiency of energy transfer depends on the distance between the donor (retinol) and the acceptor (protoporphyrin IX). Using the Förster theory, a retinolprotoporphyrin IX distance of 25 A was calculated. These results indicate that retinol and protoporphyrin IX are bound to the beta-lactoglobulin monomer at two different sites.

Serpin-like properties of alpha1-antitrypsin Portland towards furin convertase.

Recent studies have demonstrated that a serpin variant, alpha1-antitrypsin Portland (AT-PDX), can inhibit the mammalian convertase furin. Here, we examine the mechanism by which this inhibition takes place. We find that furin, which does not belong to the trypsin-like serine protease family, the usual targets of serpins, forms an SDS-heat denaturation-resistant complex with AT-PDX both in vitro and in vivo. AT-PDX inhibited furin with an association rate constant (k(ass)) of 1.5 x 10(6) M(-1) s(-1) which is similar to k(ass) values reported for serpins with trypsin-like enzymes. These results illustrate that AT can be modified to act essentially as a suicide inhibitor of furin, an enzyme of the subtilase superfamily of serine proteases.