Antibodies against cerebral M1 cholinergic muscarinic receptor from schizophrenic patients: molecular interaction

We demonstrated the presence of circulating Abs from schizophrenic patients able to interact with cerebral frontal cortex-activating muscarinic acetylcholine receptors (mAChR). Sera and purified IgG from 21 paranoid schizophrenic and 25 age-matched normal subjects were studied by indirect immunofluorescence, flow cytometry, immunoblotting, dot blot, ELISA, and radioligand competition assays. Rat cerebral frontal cortex membranes and/or a synthetic peptide, with an amino acid sequence identical with that of human M(1) mAChR, were used as Ags. By indirect immunofluorescence and flow cytometry procedures, we proved that serum-purified IgG fraction from schizophrenic patients reacted to neural cell surfaces from rat cerebral frontal cortex. The same Abs were able to inhibit the binding of the specific M(1) mAChR radioligand [(3)H]pirenzepine. Immunoblotting experiments showed that IgG from schizophrenic patients revealed a band with a molecular mass coincident to that labeled by an anti-M(1) mAChR Ab. Using synthetic peptide for dot blot and ELISA, we demonstrated that these Abs reacted against the second extracellular loop of human cerebral M(1) mAChR. Also, the corresponding affinity-purified antipeptide Ab displayed an agonistic-like activity associated to specific receptor activation, increasing cyclic GMP production and inositol phosphate accumulation, and protein kinase C translocation. This paper gave support to the participation of an autoimmune process in schizophrenia.     

Measuring microvascular density in tumors by digital dissection

OBJECTIVE: To develop and validate a digital dissection techniquefor measuring the cross-sectional area of blood vessels in histologic sections of tumors routinely stained with hematoxylin and eosin. STUDY DESIGN: The procedure was first validated in four experimental tumors in rats by comparing the results of the digital dissection technique to functional estimates of the blood volume in the tumors as measured by dynamic, contrast-enhanced magnetic resonance imaging. The method was then tested on a variety of experimental and human tumors. RESULTS: The digital dissection technique yielded results that exactly matched the functional measurements of blood volume infour experimental tumors. Digital dissection of 40 additional tumors in rats showed that 21 infiltrating ductal carcinomas had significantly greater microvascular density (MVD) than 19 benign fibroadenomas (12% vs. 7.9%, P=.028 by two-tailed t test). In 10 human breast carcinomas the MVD was consistently greater than the measurement of blood vessel density as identified by immunohistochemical staining for factor VIII. The between-run coefficients of variation for the MVD assay were 12% (n = 5) for a human breast cancer and 18% (n = 5)for an experimental rat tumor. CONCLUSION: The digital dissection technique is a reproducible, objective and accurate method of measuring MVD in sections of tumors that are routinely stained with hematoxylin and eosin.     

Rat extramedullary adipose tissue as a source of osteochondrogenic progenitor cells

Human liposuction aspirates contain pluripotent adipose-derived mesodermal stem cells that have previously been shown to differentiate into various mesodermal cell types, including osteoblasts and chondrocytes. To develop an autologous research model of bone and cartilage tissue engineering, the authors sought to determine whether rat inguinal fat pads contain a similar population of osteochondrogenic precursor cells. It was hypothesized that the rat inguinal fat pad contains adipose-derived multipotential cells that resemble human adipose-derived mesodermal stem cells in their osteochondrogenic capacity. To test this, the authors assessed the ability of cells isolated from the rat inguinal fat pad to differentiate into osteoblasts and chondrocytes by a variety of lineage-specific histologic stains.Rat inguinal fat pads were isolated and processed from Sprague-Dawley rats into a fibroblast-like cell population. Cell cultures were placed in pro-osteogenic media containing dexamethasone, ascorbic acid, and beta-glycerol phosphate. Osteogenic differentiation was assessed at 2, 4, and 6 weeks. Alkaline phosphatase activity and von Kossa staining were performed to assess osteoblastic differentiation and the production of a calcified extracellular matrix. Cell cultures were also placed in prochondrogenic conditions and media supplemented with transforming growth factor-beta1, insulin, transferrin, and ascorbic acid. Chondrogenic differentiation was assessed at 2, 7, and 14 days by the presence of positive Alcian blue staining and type II collagen immunohistochemistry. Cells placed in osteogenic conditions changed in structure to a more cuboidal shape, formed bone nodules, stained positively for alkaline phosphatase activity, and secreted calcified extracellular matrix by 2 weeks. Cells placed in chondrogenic conditions formed cartilaginous nodules within 48 hours that stained positively for Alcian blue and type II collagen. The authors identified the rat inguinal fat pad as a source of osteochondrogenic precursors and developed a straightforward technique to isolate osteochondrogenic precursors from a small animal source. This relatively easily obtained source of osteochondrogenic cells from the rat may be useful for study of tissue engineering strategies and the basic science of stem cell biology.     

Zebrafish mutants identify an essential role for laminins in notochord formation

Basement membranes are thought to be essential for organ formation, providing the scaffold on which individual cells organize to form complex tissues. Laminins are integral components of basement membranes. To understand the development of a simple vertebrate organ, we have used positional cloning to characterize grumpy and sleepy, two zebrafish loci known to control notochord formation, and find that they encode laminin beta1 and laminin gamma1, respectively. Removal of either chain results in the dramatic loss of laminin 1 staining throughout the embryo and prevents formation of the basement membrane surrounding the notochord. Notochord cells fail to differentiate and many die by apoptosis. By transplantation, we demonstrate that, for both grumpy and sleepy, notochord differentiation can be rescued by exogenous sources of the missing laminin chain, although notochordal sources are also sufficient for rescue. These results demonstrate a clear in vivo requirement for laminin beta1 and laminin gamma1 in the formation of a specific vertebrate organ and show that laminin or the laminin-dependent basement membrane is essential for the differentiation of chordamesoderm to notochord.     

4-Hydroxytamoxifen is a potent inhibitor of the mitochondrial permeability transition

The effects of 4-hydroxytamoxifen (OHTAM), the major active metabolite of the antiestrogen tamoxifen used in the breast cancer therapy, were studied on the mitochondrial permeability transition (MPT) and bioenergetic functions of mitochondria to evaluate the mechanisms underlying the cell death and toxic effects. The MPT was induced in vitro by incubating rat liver mitochondria with 1 mM inorganic phosphate plus Ca2+ and with tert-butyl hydroperoxide. OHTAM provides protection against the Ca2+-induced mitochondrial swelling, depolarization of the mitochondrial membrane potential (deltapsi), loss of electrophoretic Ca2+ uptake capacity and uncoupling of respiration, similarly to cyclosporine A. The concentrations of OHTAM used do not significantly affect deltapsi, respiratory control and adenosine diphosphate/oxygen ratios and induce repolarization and Ca2+ re-uptake, suggesting that such inhibitory effects of OHTAM were due to the prevention of the MPT induction and not due to the inhibition of the mitochondrial Ca2+ uniporter. Since the MPT induction has been linked to an oxidized shift in the mitochondrial redox state and/or increase in the generation of reactive oxygen species, the MPT prevention by OHTAM may be related to its high antioxidant capacity.     

Change in the accessibility of an epitope of the human granulocyte-colony stimulating factor after binding to receptors

In a previous work we demonstrated that monoclonal antibody (mAb) 8C2 recognized a human granulocyte-colony stimulating factor (hG-CSF) region left unmasked after binding to placenta receptors, whereas mAb 6E3 defined a receptor-buried epitope. Herein we examined the role of these antigenic regions on the proliferative response induced by hG-CSF on a myeloid leukaemia cell line. Both mAbs significantly inhibited the hG-CSF-induced cell growing, although epitope 8C2 but not 6E3 remained exposed in hG-CSF:cell receptor complexes. When cytokine:receptor complexes already formed at 4 degrees C were incubated 1 h at 37 degrees C under conditions preventing the internalization, a significant reduction in the amount of accessible 8C2 epitopes was evident. However, this effect was not observed when mAb 8C2:hG-CSF complexes previously bound to cells were incubated at 37 degrees C. Thus, results suggest that a receptor oligomerization process could account for the temperature-induced epitope 8C2 masking. The identification of epitope 8C2 accomplished by synthesis of overlapping octapeptides, revealed that it is formed by sequences 39-52 and 155-164, both in close proximity in the three-dimensional structure of the hG-CSF molecule. Since part of this region has been proposed as a second binding site to receptors, we infer that the change of epitope 8C2 accessibility could be the result of either receptor aggregation or epitope binding to another receptor. In addition, our data support the hypothesis that a ligand-induced receptor oligomerization is required for transduction of cytokine signals.     

Basic calcium phosphate crystals stimulate the endocytotic activity of cells--inhibition by anti-calcification agents

Pathological calcifications are associated with many medical conditions including diabetes, breast cancer, and crystals-associated osteoarthritis. The deposition of calcium-containing crystals on cells induces detrimental cellular effects and speeds up the progression of associated diseases. We carried out the present study to test the hypotheses that calcium-containing crystals may stimulate the influx of other molecules existing in the extracellular fluid disturbing normal molecular signaling and that anti-calcification agent will inhibit such endocytotic process. We found that basic calcium phosphate (BCP) crystals greatly stimulated the endocytotic activity of cells by rendering the cells more permeable and that the anti-calcification agent phosphocitrate and several others inhibited the crystals-mediated endocytosis. This is the first study reporting that the endocytotic activity of cells is affected by BCP crystals and that such endocytotic activity can be inhibited by anti-calcification agents. Since calcium-containing crystals are associated with many human diseases and in many circumstances are associated with apoptotic bodies, extracellular and matrix vesicles where DNA fragments, small peptides, and minerals are released into extracellular space, the findings reported here are important for our understanding of the complex biological effects and the potential pathological role of calcium-containing crystals in crystals-associated diseases, and for the development of disease modifying agents as well.     

Genetic study of Coris julis (Osteichthyes, Perciformes, Labridae) evolutionary history and dispersal abilities

Microsatellite markers have been used to study the genetic variability of rainbow wrasse (Coris julis) Mediterranean and Atlantic populations. Differentiation tests failed to reveal any significant genetic differentiation among samples from continental Portugal and the Azores, despite more than 1800 km of geographical separation. Preliminary results tended to indicate a significant genetic differentiation among Atlantic and Mediterranean samples. It also supported the specific status of Cape Verde populations (Coris atlantica). We compare these results with previous mtDNA analyses and propose a biogeographic scenario that could explain our results.     

Photoactivation of phthalocyanine-loaded low density lipoproteins induces a local oxidative stress that propagates to human erythrocytes: protection by caffeic acid

Toxic effects imposed to human erythrocytes by low density lipoproteins carrying phthalocyanines used in photodynamic therapy (PDT) of tumors are described. This study was aimed at evaluating cytotoxic effects induced by reactive species produced locally in photosensitizer-loaded lipoproteins and further transferred to the cells. The experimental set up designed to examine these interactions starts with the loading of human plasma with the photosensitizer, the subsequent rapid purification and dialysis of the LDL fraction and incubation with human erythrocytes. This experimental model was assessed by following leakage of endogenous K+ from cells, electrochemical detection of oxygen, spectroscopic determination of conjugated dienes, phthalocyanine, SH groups and hemoglobin, analysis of fatty acids by gas chromatography and identification of a-tocopherol by HPLC. Photosensitizer-loaded lipoproteins become more susceptible to oxidation, exhibiting shorter lag phases of lipid oxidation, higher rates of oxidation and increased loss of endogenous alpha-tocopherol when challenged with peroxyl radicals and copper, as compared with native lipoproteins from the same plasma sample. Incubation of photosensitized lipoproteins with erythrocytes under light (>560 nm) results in a sigmoidal efflux of K+ followed by hemolysis. The phenolic antioxidant caffeic acid inhibits lipoprotein oxidation induced by peroxyl radicals, either in native or photosensitizer-loaded fractions, delays hemolysis of erythrocytes and partially prevents membrane loss of SH groups in ghosts, but not the efflux of K+. Mechanistically, a chain lipid peroxidation reaction does not participate in the toxic effects to cells but a specific pool of membrane SH groups sensitive to caffeic acid is likely to be involved. This study suggests that an oxidative stress occurring locally in phthalocyanine-loaded low density lipoproteins may further induce cytotoxic effects by targeting specific SH groups at the cell membrane level. The physiological relevance of these findings and the beneficial use of antioxidants are discussed in the context of PDT.     

The interaction of resveratrol with ferrylmyoglobin and peroxynitrite; protection against LDL oxidation

Resveratrol (3,4',5-trihydroxystilbene) is a natural phytoalexin synthesized in response to injury or fungal attack, found in the grape skin and wine, specially red wine. A large number of studies have demonstrated that resveratrol regulates many biological activities, namely protection against atherosclerosis by a set of pharmacological properties, including the antioxidant activity. In this study, we explored the capacity of resveratrol in protecting low density lipoproteins (LDL) against either ferrylmyoglobin- or peroxynitrite-mediated oxidation and the underlying mechanisms of its antioxidant potential. Resveratrol efficiently decreases the accumulation of hydroperoxides in LDL promoted by ferrylmyoglobin, a potent oxidant formed by the reaction of metmyoglobin with hydrogen peroxide, in a concentration-dependent manner, promptly reducing the oxoferryl complex to metmyoglobin. Simultaneously, resveratrol is consumed as detected by the rapid decrease in the characteristic peak at 310 nm, in a similar way to that observed upon its reaction with peroxidase/H 2 O 2 , pointing to a mechanism of one-electron oxidation and subsequent resveratrol dimer formation. On the other hand, resveratrol inhibits LDL apoprotein modifications induced by peroxynitrite, another potent oxidant formed by the reaction between superoxide and nitric oxide, as assessed by the decrease in apo-B net charge alterations and in carbonyl groups formation mediated by that oxidant. Resveratrol also interacts with peroxynitrite in a similar way to that observed with laccases, suggesting a mechanism of resveratrol oxidation rather than a nitration one. These mechanisms are discussed. Considering that either ferrylmyoglobin or peroxynitrite are physiologically relevant oxidants implicated in several pathologies, including atherosclerosis, our results certainly contribute to the understanding of the antioxidant action of resveratrol and consequently provide a new approach for the cardiovascular benefits associated with moderate consumption of red wine.