Effect of pasteurization and storage on some components of pooled human milk

Pooled human milk was subjected to Holder pasteurization and storage at −20°C up to 90 days and examined for its content of fat and

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-lactate and for lipid composition. This treatment reduced fats by 6% and

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-lactate by at least 7%. In addition, pasteurization and storage induced triglyceride hydrolysis. The absolute amount of free fatty acids (FFAs) which was 0.5% after collection, doubled after pasteurization and rose even more after storage. Different FFA compositions were found by several authors using the same analytical method even for milk samples subjected to the same treatment. More detailed information on procedures must be given to explain the different results.     

Evolution: an axiomatic perspective.

In this paper we present an axiomatic theory of evolution which is inspired by the reading of a paper written by G.V. Schiaparelli in 1898. Schiaparelli was a famous astronomer, but he also studied the Darwinian ideas. We propose five axioms which can characterize the theory of evolution. We have also written these axioms using the language of the logic of predicates of first order with some constant monadic and dyadic predicates and appropriate functionals. But we can use other types of logic. So we can examine the concepts of species and of speciation. Then we introduce the interesting notion of generation distance. Moreover we give a theorem which establishes a geometrical model of our theory. If we analyze further the fourth axiom (which concerns the notion of generation distance) we can propose an elementary dynamical model by which we can represent possible evolutionary dynamics. These dynamics partially depend on random quantities.     

Conformational analysis of the Galpha(s) protein C-terminal region.

The C-terminal domain of the heterotrimeric G protein a-subunits plays a key role in selective activation of G proteins by their cognate receptors. Several C-terminal fragments of Galpha(s) (from 11 to 21 residues) were recently synthesized. The ability of these peptides to stimulate agonist binding was found to be related to their size. Galpha(s)(380-394) is a 15-mer peptide of intermediate length among those synthesized and tested that displays a biological activity surprisingly weak compared with that of the corresponding 21-mer peptide, shown to be the most active. In the present investigation, Galpha(s)(380-394) was subjected to a conformational NMR analysis in a fluorinated isotropic environment. An NMR structure, calculated on the basis of the data derived from conventional 1D and 2D homonuclear experiments, shows that the C-terminal residues of Galpha(s)(380-394) are involved in a helical arrangement whose length is comparable to that of the most active 21 -mer peptide. A comparative structural refinement of the NMR structures of Galpha(s)(380-394) and Galpha(s)(374-394)C379A was performed using molecular dynamics calculations. The results give structural elements to interpret the role played by both the backbone conformation and the side chain arrangement in determining the activity of the G protein C-terminal fragments. The orientation of the side chains allows the peptides to assume contacts crucial for the G protein/receptor interaction. In the 15-mer peptide the lack as well as the disorder of some N-terminal residues could explain the low biological activity observed.     

The influence of asymmetric carbon atoms of the side chains on the conformational properties of polypeptides: Comparison of diastereomeric homooligopeptides of L-isoleucine and D-alloisoleucine

The conventionally protected oligopeptides of the two homologous series Boc-(L -Ile)_n-OMe and Boc-(D -aIle)_n-OMe (n = 2–6) were synthesized in a standard stepwise fashion and their uv and CD spectra in 2,2,2-trifluoroethanol, and solid-state ir spectra were investigated. In addition, two oligomeric products derived from the NCAs of L -isoleucine and of D -allo-isoleucine and having a DP of 20 and 12, respectively, were studied in the solid state by x-ray and ir. No substantial differences between the properties of the diastereomeric oligomers in the solid state were noticed, a β-structure being very likely at least for the Boc-protected hexapeptides and the higher oligomers. In contrast, differences were observed between the spectroscopic properties of the diastereomeric oligopeptides, and especially of the hexapeptides, in trifluoroethanol solution. The different properties of the hexapeptides in solution were related to the existence, in the case of Boc-(L -Ile)₆-OMe, of soluble molecular aggregates in which the peptide chains assume the β-conformation. These results provide an additional example of the influence of the configuration of asymmetric carbon atoms of the side chains on the conformational properties of peptide molecules in solution.     

Exploring interaction of beta-amyloid segment (25-35) with membrane models through paramagnetic probes.

The accumulation of beta-amyloid peptides into senile plaques is one of the hallmarks of Alzheimer's disease (AD). There is mounting evidence that the lipid matrix of neuronal cell membranes plays an important role in the beta-sheet oligomerization process of beta-amyloid. Abeta(25-35), the sequence of which is GSNKGAIIGLM, is a highly toxic segment of amyloid beta (Abeta)-peptides, which forms fibrillary aggregates. In the present work, two spin-labelled Abeta(25-35) analogues containing the nitroxide group of the amino acid TOAC (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) as a paramagnetic probe at the N- or the C-terminus of the peptide sequence, respectively, were synthesized in order to investigate the peptide-membrane interaction. The orientation and associated changes of the peptide conformation in the presence of different artificial membrane models (micelles, liposomes) were evaluated by electron paramagnetic resonance and circular dichroism techniques. The results of this study allowed us to propose a model in which the C-terminal portion of the peptide is highly associated to the membrane, while the N-terminal part extends into the aqueous phase with occasional contacts with the lipid head-group region. Interestingly, the interaction of the C-terminal portion of the peptide is particularly enhanced in the presence of sodium dodecyl sulfate (SDS) molecules.     

Fluorescent, internally quenched, peptides for exploring the pH-dependent substrate specificity of cathepsin B.

Cathepsin B is a cysteine protease that in tumor tissues is localized in both acidic lysosomes and extracellular spaces. It can catalyze the cleavage of peptide bonds by two mechanisms: endoproteolytic attack with a pH optimum around 7.4, and attack from the C-terminus with a pH optimum at 4.5-5.5. In this work, seven fluorescent, internally quenched, decapeptides have been synthesized using the prototypical cathepsin B selective substrate Z-Phe-Arg-AMC as a lead, and used to identify the structural factors determining the susceptibility of peptides to hydrolysis at acidic and neutral pH values. Each peptide differs from the others in one amino acid (residue 6) and contains a highly fluorescent Nma group linked to the alpha-amino function of the N-terminal Orn residue and a Dnp group linked to the side chain of the Lys(8) residue acting as a quencher. Proteolytic cleavage was monitored by measuring the increase of fluorescence at 440 nm upon excitation at 340 nm, and the cleavage sites were determined by HPLC followed by ESI-MS analysis. Peptides containing Ala or Phe at position 6 are good substrates for the enzyme at both pH 5.0 and 7.4. By contrast, those containing Glu, Asp, Lys or Val are not cleaved at all by cathepsin B at pH 7.4, and are poorly hydrolyzed at pH 5.0. These findings provide new information for the rational design of cathepsin B-activated peptide-containing anticancer drugs.     

Activation of the IGF1 system characterizes cholangiocyte survival during progression of primary biliary cirrhosis.

We evaluated the IGF1 system in cholangiocytes of primay biliary cirrhosis (PBC) patients and investigated the relationships with apoptosis. Biopsies of PBC patients (n=32) and normal subjects (n=5) were investigated by immunohistochemistry for expression in cholangiocytes of IGF1, IGF1-R, pAKT, terminal deoxynucleotide transferase end labeling (TUNEL), Bax (proapoptotic protein), and Bcl2 (antiapoptotic protein). Whereas normal cholangiocytes were almost negative, cholangiocytes of PBC patients showed strong IHC staining for IGF1, IGF1-R, and pAKT, which increases from stage I to stage IV, where >70% of cholangiocytes were positive. Bax/Bcl2 ratio reached the highest value (4.6) in PBC stage III when apoptosis is maximal (24% TUNEL positivity), whereas it declines in stage IV (1.4) when only 7.8% cholangiocytes were TUNEL positive. In PBC stages III and IV, expression of IGF1, IGF1-R, and pAKT in cholangiocytes was directly correlated with the antiapoptotic Bcl2 and inversely correlated with proapoptotic Bax, Bax/Bcl2 ratio, and TUNEL positivity. In conclusion, cholangiocytes of PBC patients showed a marked increase in IGF1, IGF1-R, and pAKT expression involving most cholangiocytes surviving in the terminal ductopenic stage. This was associated and correlated with a balance of pro- and antiapoptotic proteins favoring survival rather than apoptosis, suggesting a major role of IGF1 system in promoting cholangiocyte survival.