Vascular enumeration as a significant prognosticator for invasive breast carcinoma

PURPOSE: Vascular enumeration has been claimed to be an independent prognosticator for invasive breast cancer. Most of the studies have performed a manual count of the vessels. Few investigators have used image analyzers to reduce subjectivity in the measures. The aim of this retrospective study was to compare the manual vessel count to the counts obtained with an image analyzer and to estimate their possible prognostic influence. MATERIALS AND METHODS: We selected 112 patients with invasive ductal carcinoma, treated with radical mastectomy followed by chemotherapy and with a minimal follow-up time of 60 months (5 years). After immunostaining with factor VIII, we performed a double count of the vessels. First, we performed a manual count following Gasparini's criteria, and second, we used an image analyzer (Microm, Barcelona, Spain) to count the vessels in eight adjacent microscopic fields with a 20x objective, to reach a total area of 0.98 mm2. The image analyzer calculated the total number of vessels, their size and also the percentage of the field occupied by the vessels. RESULTS: In our series, vascular enumeration showed no significant association with histologic grade (Bloom-Scarff-Richardson grading), tumor size, or staging. CONCLUSION: Vascular enumeration with both methods was an independent prognosticator for relapse-free-survival (RFS) in both node-negative and -positive patients in the univariate analysis, but only vascular enumeration with the image analyzer was an independent prognostic factor in the multivariate analysis, together with lymph node metastases.     

Immunohistochemical expression of p53 and c-erbB-2 in breast carcinoma: relation with epidemiologic factors, histologic features and prognosis

We have performed immunohistochemical staining for p53 and c-erbB-2 on formaldehyde-fixed, paraffin-embedded primary invasive ductal carcinomas from 112 patients, with a minimal follow-up time of 60 months. All of them had received postoperative chemoradiation therapy. We have analyzed the association of these factors with epidemiologic risk factors, histopathologic features and hormonal receptor status and the influence on prognosis. Our results indicate that the expression of c-erbB-2 protein defines a group of node-negative patients with poor prognosis. The overexpression of c-erbB-2 has shown a significant association with estrogen receptor status (those tumors expressing c-erbB-2 are usually estrogen receptor negative), presence of fibrosis and lymphoplasmacytoid infiltrates. P53 expression has shown no relation either with prognosis or with any other histopathologic or clinical feature. The only factors with prognostic influence in our series have been tumor size, the presence of node metastases, TNM stage and the prognostic morphometric index (Baak's index), apart from c-erbB-2 in node-negative patients. However, only the TNM stage showed an independent association with prognosis after a multivariate analysis. In summary, in our experience the expression of p53 protein has no prognostic influence on breast carcinoma, and TNM stage remains to be as the most powerful prognostic factor in these patients.     

In vitro cytotoxicity of guanylhydrazones (MGBG and PGBG) on cultured Chinese hamster ovary cells

Periosteum was obtained within 10 days of injury from the site of 17 adult tibial diaphyseal fractures during internal fixation. Osteogenic cells, non-osteogenic cells and vascular elements were identified in situ using a variety of techniques. In all cases, the periosteum was thickened with randomly distributed plaques of cartilage and bone. Cells covering newly formed bone trabeculae expressed osteocalcin. Lectin-binding revealed high vascularity. Few mast cells were observed. Macrophages and acid phosphatase positive cells, some multinucleate, were observed in abundance. These findings suggest that the repair of the adult human diaphyseal fracture is similar to that of experimental fractures in rapidity of onset, high vascularity and in bone and cartilage formation. They differ in the fact that chondrogenesis and osteogenesis appear to be simultaneous in human fractures but sequential in experimental fractures. The paucity of mast cells suggests that they probably play no significant role in the repair of the human fractures.     

Antioxidant Systems from Pepper (Capsicum annuum L.): Involvement in the Response to Temperature Changes in Ripe Fruits

Sweet pepper is susceptible to changes in the environmental conditions, especially temperatures below 15 °C. In this work, two sets of pepper fruits (Capsicum annuum L.) which underwent distinct temperature profiles in planta were investigated. Accordingly, two harvesting times corresponding to each set were established: Harvest 1, whose fruits developed and ripened at 14.9 °C as average temperature; and Harvest 2, with average temperature of 12.4 °C. The oxidative metabolism was analyzed in all fruits. Although total ascorbate content did not vary between Harvests, a shift from the reduced to the oxidized form (dehydroascorbate), accompanied by a higher ascorbate peroxidase activity, was observed in Harvest 2 with respect to Harvest 1. Moreover, a decrease of the ascorbate-generating enzymatic system, the γ-galactono-lactone dehydrogenase, was found at Harvest 2. The activity values of the NADP-dependent dehydrogenases analyzed seem to indicate that a lower NADPH synthesis may occur in fruits which underwent lower temperature conditions. In spite of the important changes observed in the oxidative metabolism in fruits subjected to lower temperature, no oxidative stress appears to occur, as indicated by the lipid peroxidation and protein oxidation profiles. Thus, the antioxidative systems of pepper fruits seem to be involved in the response against temperature changes.     

Enzymatic Synthesis of α‐Glucosides of Resveratrol with Surfactant Activity

We report the synthesis of a series of α‐glucosyl derivatives of resveratrol (3,5,4′‐trihydroxystilbene) by a transglycosylation reaction catalyzed by the enzyme cyclodextrin glucanotransferase (CGTase) using starch as glucosyl donor. Several reaction parameters (temperature, solvent composition, enzyme concentration and starch/resveratrol ratio) were optimized. The yield of α‐glucosylated products reached 50% in 24 h. The structures of the derivatives were determined by a combination of amyloglucosidase‐hydrolysis tests, MS and 2D‐NMR. Three families of products were obtained: glucosylated at 3‐OH, at 4′‐OH and at both 3‐OH and 4′‐OH. The bonds between glucoses were basically α(1→4). Interestingly, the water solubilities of the α‐glucosylated derivatives were at least 65‐ and 5‐fold higher than those of resveratrol and the natural β‐glucosylated derivative (piceid), respectively. In contrast with piceid, the synthesized α‐glucosylated compounds exhibited surfactant activity, with critical micelle concentration (CMC) values in the range 0.5–3.6 mM. Although the incorporation of a glucosyl moiety caused a loss of antioxidant activity (more pronounced in the position 3‐OH compared with 4′‐OH), the fact that the glycosides need to be converted into the aglycones before they are absorbed minimizes such an effect. In contrast, the modification of physicochemical properties such as solubility and partition coefficient by glycosylation could exert a positive influence on the bioavailability of resveratrol.     

Application of a microassay method to study enzymatic hydrolysis of pretreated wheat straw

BACKGROUND: The conversion of lignocellulosic biomass to ethanol includes a disruptive pretreatment process followed by enzyme‐catalyzed hydrolysis of the cellulose and hemicellulose components to fermentable sugars. As the cost and hydrolytic efficiency of enzymes are major factors that restrict the commercialization of biomass conversion processes, significant efforts are made nowadays to improve the enzymatic mixtures and make the process cost‐effective. RESULTS: In this work, enzymatic microassays have been developed and validated to test new different enzymatic formulations on real lignocellulosic substrates. Homogeneous handsheets from steam pretreated wheat straw were elaborated to be used as substrate. The microassay was adapted to test both water‐insoluble solids and the whole slurry as substrates. Results in hydrolysis microassays were comparable with those obtained in standard flask assays using pretreated wheat straw. Moreover, using the enzymatic microassays, two novel preparations have been evaluated, demonstrating the ability of microassays to discriminate between different enzymatic mixtures. CONCLUSIONS: This enzymatic microassay represents a rapid method to test the performance of new selected cellulase enzymes on real pretreated lignocellulosic substrates. This microassay will enable evaluation of enzyme components separately, or optimized mixtures. Copyright © 2010 Society of Chemical Industry     

Drebrin Regulates Acetylcholine Receptor Clustering and Organization of Microtubules at the Postsynaptic Machinery

Proper muscle function depends on the neuromuscular junctions (NMJs), which mature postnatally to complex “pretzel-like” structures, allowing for effective synaptic transmission. Postsynaptic acetylcholine receptors (AChRs) at NMJs are anchored in the actin cytoskeleton and clustered by the scaffold protein rapsyn, recruiting various actin-organizing proteins. Mechanisms driving the maturation of the postsynaptic machinery and regulating rapsyn interactions with the cytoskeleton are still poorly understood. Drebrin is an actin and microtubule cross-linker essential for the functioning of the synapses in the brain, but its role at NMJs remains elusive. We used immunohistochemistry, RNA interference, drebrin inhibitor 3,5-bis-trifluoromethyl pyrazole (BTP2) and co-immunopreciptation to explore the role of this protein at the postsynaptic machinery. We identify drebrin as a postsynaptic protein colocalizing with the AChRs both in vitro and in vivo. We also show that drebrin is enriched at synaptic podosomes. Downregulation of drebrin or blocking its interaction with actin in cultured myotubes impairs the organization of AChR clusters and the cluster-associated microtubule network. Finally, we demonstrate that drebrin interacts with rapsyn and a drebrin interactor, plus-end-tracking protein EB3. Our results reveal an interplay between drebrin and cluster-stabilizing machinery involving rapsyn, actin cytoskeleton, and microtubules.     

SMA-10 Is a Non-Canonical Member of the TGF-β Sma/Mab Pathway and Immunity Regulator via the DAF-2 Insulin Receptor in Caenorhabditis elegans

Transforming growth factor β (TGF-β) signalling pathways are highly conserved across metazoa and play essential roles not only during development but also in adult tissue maintenance. Alterations of these pathways usually result in a plethora of pathologies. In the nematode Caenorhabditis elegans, the TGF-β Sma/Mab (small/male abnormal) pathway regulates various worm phenotypes such as body size, immune response, ageing, matricide and reproductive span. SMA-10 has been described as a positive modulator of worm body size through the TGF-β Sma/Mab pathway. To better understand if SMA-10 is a core component of the pathway, we use gene epistatic analysis to assess the contribution of SMA-10 to various phenotypes regulated by TGF-β Sma/Mab. We confirm that SMA-10 controls body size and find that it also affects the matricide and reproductive span of the nematodes. However, neither male tail formation (previously reported) nor ageing appeared altered. Lastly, although null sma-10 worms are more susceptible to Pseudomonas aeruginosa infections than wild-types, this response does not depend on TGF-β Sma/Mab but on the insulin receptor DAF-2. We also show that the expression of sma-10 in either hypodermis or intestine fully rescues the wild-type immune response. Our results contribute to understanding the role of SMA-10 as a context-dependent component of TGF-β Sma/Mab, and reveal a function of SMA-10 in immunity in association to the Insulin/insulin-like growth factor signalling (IIS) pathway.