Secretome compartment is a valuable source of biomarkers for cancer-relevant pathways

In principle, targeted therapies have optimal activity against a specific subset of tumors that depend upon the targeted molecule or pathway for growth, survival, or metastasis. Consequently, it is important in drug development and clinical practice to have predictive biomarkers that can reliably identify patients who will benefit from a given therapy. We analyzed tumor cell-line secretomes (conditioned cell media) to look for predictive biomarkers; secretomes represent a potential source for potential biomarkers that are expressed in intracellular signaling and therefore may reflect changes induced by targeted therapy. Using Gene Ontology, we classified by function the secretome proteins of 12 tumor cell lines of different histotypes. Representations and hierarchical relationships among the functional groups differed among the cell lines. Using bioinformatics tools, we identified proteins involved in intracellular signaling pathways. For example, we found that secretome proteins related to TGF-beta signaling in thyroid cancer cells, such as vasorin, CD109, and βIG-H3 (TGFBI), were sensitive to RPI-1 and dasatinib treatments, which have been previously demonstrated to be effective in blocking cell proliferation. The secretome may be a valuable source of potential biomarkers for detecting cancer and measuring the effectiveness of cancer therapies.     

Protein Kinase C ε Expression in Platelets from Patients with Acute Myocardial Infarction

Objective

Platelets play crucial roles in the pathophysiology of thrombosis and myocardial infarction. Protein kinase C ε (PKCε) is virtually absent in human platelets and its expression is precisely regulated during human megakaryocytic differentiation. On the basis of what is known on the role of platelet PKCε in other species, we hypothesized that platelets from myocardial infarction patients might ectopically express PKCε with a pathophysiological role in the disease.

Methods and Results

We therefore studied platelet PKCε expression from 24 patients with myocardial infarction, 24 patients with stable coronary artery disease and 24 healthy subjects. Indeed, platelets from myocardial infarction patients expressed PKCε with a significant frequency as compared to both stable coronary artery disease and healthy subjects. PKCε returned negative during patient follow-up. The forced expression of PKCε in normal donor platelets significantly increased their response to adenosine diphosphate-induced activation and adhesion to subendothelial collagen.

Conclusions

Our data suggest that platelet generations produced before the acute event retain PKCε-mRNA that is not down-regulated during terminal megakaryocyte differentiation. Results are discussed in the perspective of peri-infarctual megakaryocytopoiesis as a critical component of myocardial infarction pathophysiology.     

Bovine lactoferrin in preventing preterm delivery associated with sterile inflammation

Preterm delivery (PTD) occurs before the 37th week of gestation. Iron deficiency anemia and inflammatory processes either related to infection or sterile inflammatory response represent risk factors for PTD. Bovine lactoferrin (bLf), an emerging important regulator of iron and inflammatory homeostasis, can represent a new therapeutic approach for PTD treatment. Here an open-label cohort and subcohort study is reported. The cohort was designed to assess the effect of bLf oral administration on iron and inflammatory homeostasis in anemic pregnant women. The subcohort including women of the cohort with PTD threat was additionally treated with bLf intravaginal administration. A significant improvement of hematological parameters was observed in the women's cohort together with a consistent decrease of serum interleukin-6 (IL-6) levels. Combined administration of oral and intravaginal bLf to the women's subcohort with PTD threat decreased IL-6 in both serum and cervicovaginal fluids, cervicovaginal prostaglandin F(2α), and suppressed uterine contractility. BLf administration blocked further shortening of cervical length and the increase of fetal fibronectin thus prolonging the length of pregnancy. The deliveries occurred between the 37th and 38th week of gestation. These results provide strong evidence for a role of bLf in PTD treatment, thus extending the therapeutic potential of this multifunctional natural protein.     

Ca2+ binding to bovine lactoferrin enhances protein stability and influences the release of bacterial lipopolysaccharide.

Bovine lactoferrin (bLf) is known to damage the outer membrane of Gram-negative bacteria by binding to bacterial lipopolysaccharide (LPS). We report that LPS is released from bacterial outer membranes also when apo- or metal-saturated Lf is separated from bacterial cells by a dialysis membrane. This process occurs in phosphate-buffered saline with no added Ca2+ and Mg2+ and is hindered by addition of these cations. The effect of bLf is similar to that induced by EDTA and has been ascribed to chelation of Ca2+. In fact, it may be envisaged that Ca2+-binding sites on LPS have different affinities and that bLf can remove those ions that are more weakly bound. Ca2+ binding does not alter Lf iron-binding properties significantly or its UV and CD spectral features but brings about changes in the FT-IR bands due to carboxylate residues. Ca2+ binding is characterized by an apparent dissociation constant of 6 microM and a stoichiometry of 1.55 Ca2+ per Lf molecule; it enhances bLf stability towards chemical and thermal denaturation. The increase in stability takes place in both the apo- and iron-saturated forms but not in the desialilated protein, indicating that the carboxylate groups of the sialic acid residues present on two of the glycan chains are involved in Ca2+ binding.     

Transfer of chirality by dithiophosphate ligands and chiral discrimination in the stereoselective formation of square-planar Ni(II) complexes

In the formation reaction of Ni(2+) with the chiral racemic ligand, (R)(R)bdtp(-)/(S)(S)bdtp(-), bdtp(-) = [SSPOCH)CH(3))CH(CH(3))O](-), cyclo- O,O'-[1,2-dimethylethylene] dithiophosphato ion, the meso-complex Ni[(R)(R)(lambda)bdtp][(S)(S)(delta)-bdtp] is stereoselectively produced. The meso-complex was compared with the enantiopure crystals of (+)(589)Ni[(R)(R)(lambda)bdtp](2) or (-)(589)Ni[(S)(S)(delta)bdtp](2), as well as racemic crystals, rac-(+/-)Ni[bdtp](2), which were prepared from the solution containing the two enantiomers in a 1:1 ratio. Dissociation constants in solutions indicate different stability of the meso and enantiopure complexes depending on the solvent, whereas a more efficient crystal packing, weak H-bonding, and nonbonding interactions contribute to stabilization of the meso-species over the racemic one. Molecular structures show that the outer five-membered ligand ring adopts the half-chair conformation C(2) with either the lambda or the delta chirality and the methyl groups are in equatorial (e) positions. Enantiopure ligands of (+)(589)Ni[(R)(R)(lambda)bdtp](2) and (-)(589)Ni[(S)(S)(delta)bdtp](2) induce chirality into the symmetric SSNiSS chromophore with slightly helical distortion. Thus, their CD spectra exhibit weak negative or positive Cotton effects at 662 nm. CD spectra in L(+)- and D(-)diethyltartrate of the meso-complex and racemic crystal, rac-(+/-)Ni[bdtp](2), exhibit different weak Cotton effects of opposite sign. Complexes dissociate in methanol; rac-(+/-)Ni[bdtp](2) in methanol undergoes a crystallization-induced second-order asymmetric transformation which finally yields crystals of the meso-Ni[(R)(R)(lambda)bdtp][(S)(S)(delta)bdtp] complex.     

P62 deficiency shifts mesenchymal/stromal stem cell commitment toward adipogenesis and disrupts bone marrow homeostasis in aged mice

With advancing age have been observed bone and bone marrow phenotypic alterations due to the impaired bone tissue homeostatic features, involving bone remodeling, and bone marrow niche ontogeny. The complex “inflamm-aging” pathological scenario that culminates with osteopenia and mesenchymal/stromal and hematopoietic stem cell commitment breakdown, is controlled by cellular and molecular intramural components comprising adapter proteins such as the sequestosome 1 (p62/SQSTM1). p62, a “multiway function” protein, has been reported as an effective anti-inflammatory, bone-building factor. In this view, we considered for the first time the involvement of p62 in aging bone and bone marrow of 1 year and 2 years p62^−/− mice. Interestingly, p62 deficiency provoked accelerated osteopenia and impaired niche operational activities within the bone marrow. The above findings unearthed the importance of p62 in mesenchymal stem cell maintenance/differentiation schedule in old animals and provide, at least in part, a mechanistic scenario of p62 action.     

Thermosensitive hybrid hyaluronan/p(HPMAm-lac)-PEG hydrogels enhance cartilage regeneration in a mouse model of osteoarthritis

Osteoarthritis (OA), due to cartilage degeneration, is one of the leading causes of disability worldwide. Currently, there are not efficacious therapies to reverse cartilage degeneration. In this study we evaluated the potential of hybrid hydrogels, composed of a biodegradable and thermosensitive triblock copolymer cross-linked via Michael addition to thiolated hyaluronic acid, in contrasting inflammatory processes underlying OA. Hydrogels composed of different w/w % concentrations of hyaluronan were investigated for their degradation behavior and capacity to release the polysaccharide in a sustained fashion. It was found that hyaluronic acid was controllably released during network degradation with a zero-order release kinetics, and the release rate depended on cross-link density and degradation kinetics of the hydrogels. When locally administered in vivo in an OA mouse model, the hydrogels demonstrated the ability to restore, to some extent, bone remineralization, proteoglycan production, levels of Sox-9 and Runx-2. Furthermore, the downregulation of proinflammatory mediators, such as TNF-α, NFkB, and RANKL and proinflammatory cytokines was observed. In summary, the investigated hydrogel technology represents an ideal candidate for the potential encapsulation and release of drugs relevant in the field of OA. In this context, the hydrogel matrix could act in synergy with the drug, in reversing phenomena of inflammation, cartilage disruption, and bone demineralization associated with OA.     

Histological changes during development of the cerebellum in the chick embryo exposed to a static magnetic field

Few studies have been performed to evaluate the ultrastructural changes that exposure to static magnetic fields (SMF) can cause to the processes of cell migration and differentiation in the cerebellum during development. Thus, we have studied the development of the cerebellum in the chick embryo (n = 144) under a uniform SMF (20 mT). All of our observations were done on folium VIc of Larsell's classification. The cerebella of chick embryos, which were exposed solely on day 6 of incubation and sacrificed at day 13 of incubation [short exposure (S)1; n = 24], showed an external granular layer (EGL) that was less dense than the EGL in the control group (n = 24). The molecular layer (ML) exhibited a low number of migratory neuroblastic elements. Moreover, the internal granular layer (IGL) was immature, with the cellular elements less abundant and more dispersed than in controls. In chick embryos exposed on day 6 of incubation and sacrificed at day 17 (S2; n = 24), the outstanding feature was the regeneration of the different layers of the cerebellar cortex. The cerebellar cortex of chick embryos exposed continuously to an identical field from the beginning of the incubation up to day 13 [long exposure (L)1; n = 24] or day 17 (L2; n = 24) of incubation showed a higher number of alterations than that of group S1. Electron microscopy confirmed the findings from light microscopy and, at the same time, showed clear signs of cell degeneration and delay in the process of neuronal differentiation. This was more apparent in groups L1 (100%) and L2 (100%) than in groups S1 (95.4%) and S2 (65.2%). In conclusion, the present study showed that SMF can induce irreversible developmental effects on the processes of cell migration and differentiation of the chick cerebellar cortex. Bioelectromagnetics 18:36–46, 1997. © 1997 Wiley-Liss, Inc.     

The HMG Protein T160 Colocalizes with DNA Replication Foci and Is Down-regulated during Cell Differentiation

The high mobility group protein T160, the murine homolog of the human structure-specific recognition protein 1, was first supposed to be involved in the process of V-(D)-J recombination, since it could bind to recombination signal sequence probes. We have recently cloned T160 by using an unrelated DNA probe and shown that it binds to either cruciform or linear DNA with no sequence specificity. In this work, we performed a detailed analysis of T160 expression and immunolocalization. We show that T160 is a phosphoprotein broadly conserved from yeast to mammals, with a high level of expression in all the cell lines tested and in tissues containing a high degree of proliferating cells. Indirect immunofluorescence analysis by confocal laser microscopy revealed that T160 distribution in the cell nucleus is not uniform, and focus-like staining was observed. Cell cycle studies by BrdU incorporation suggest that the appearance of T160 nuclear foci is specific of mid to late S phase. Furthermore, while T160 expression does not change during the cell cycle, it is dramatically down-regulated when cells begin to differentiate, as highlighted in C2C12 myoblasts and myotubes. The disappearance of T160 nuclear staining in multinucleated myotubes is shown. Taken together, these data suggest that its function may be less specific than V-(D)-J recombination and more related to some cellular basic process, such as DNA replication or repair.