Mesenterial filaments of the black coral <Emphasis Type="Italic">Cirrhipathes</Emphasis> cfr. <Emphasis Type="Italic">anguina</Emphasis> provide a home to developing nauplii

Inspection of two female colonies of the monopodial black coral Cirrhipathes cfr. anguina from the coral reef of the Marine Park of Bunaken (Indonesia) revealed the occurrence of crustacean developing eggs within the mesenterial filaments of the polyps. Egg diameter, which in the smallest gametes was about 50–60 μm, increased in tandem with embryo development, reaching the value of 170 μm, at the nauplius stage. The attribution to the crustacean taxon was derived from morphological investigations carried out in light and electron microscopy (TEM, SEM) on the eggs and on the embryos removed from them. The final stage of nauplius was characterised by three pairs of appendages: uniramouse antennulae, biramouse antennae and manidibulae. In addition, naupliar eye and caudal setae were also evident. These nauplii were ascribed to the larval stage of an unidentified species. Coral/copepod association could represent a reproductive strategy, put into action by some marine copepods. Incubation within an appropriate host prevents predation by planktotrophic organisms, thus reducing population depletion.     

Potential role of HER2-overexpressing exosomes in countering trastuzumab-based therapy

Exosomes are endosome-derived nanovesicles actively released into the extracellular environment and biological fluids, both under physiological and pathological conditions, by different cell types. We characterized exosomes constitutively secreted by HER2-overexpressing breast carcinoma cell lines and analyzed in vitro and in vivo their potential role in interfering with the therapeutic activity of the humanized antibody Trastuzumab and the dual tyrosine kinase inhibitor (TKI) Lapatinib anti-HER2 biodrugs. We show that exosomes released by the HER2-overexpressing tumor cell lines SKBR3 and BT474 express a full-length HER2 molecule that is also activated, although to a lesser extent than in the originating cells. Release of these exosomes was significantly modulated by the growth factors EGF and heregulin, two of the known HER2 receptor-activating ligands and naturally present in the surrounding tumor microenvironment. Exosomes secreted either in HER2-positive tumor cell-conditioned supernatants or in breast cancer patients' serum bound to Trastuzumab. Functional assays revealed that both xenogeneic and autologous HER2-positive nanovesicles, but not HER2-negative ones, inhibited Trastuzumab activity on SKBR3 cell proliferation. By contrast, Lapatinib activity on SKBR3 cell proliferation was unaffected by the presence of autologous exosomes. Together, these findings point to the role of HER2-positive exosomes in modulating sensitivity to Trastuzumab, and, consequently, to HER2-driven tumor aggressiveness.     

Methane production by treating vinasses from hydrous ethanol using a modified UASB reactor

Background

A modified laboratory-scale upflow anaerobic sludge blanket (UASB) reactor was used to obtain methane by treating hydrous ethanol vinasse. Vinasses or stillage are waste materials with high organic loads, and a complex composition resulting from the process of alcohol distillation. They must initially be treated with anaerobic processes due to their high organic loads. Vinasses can be considered multipurpose waste for energy recovery and once treated they can be used in agriculture without the risk of polluting soil, underground water or crops. In this sense, treatment of vinasse combines the elimination of organic waste with the formation of methane. Biogas is considered as a promising renewable energy source. The aim of this study was to determine the optimum organic loading rate for operating a modified UASB reactor to treat vinasse generated in the production of hydrous ethanol from sugar cane molasses.

Results

The study showed that chemical oxygen demand (COD) removal efficiency was 69% at an optimum organic loading rate (OLR) of 17.05 kg COD/m³-day, achieving a methane yield of 0.263 m³/kg COD_added and a biogas methane content of 84%. During this stage, effluent characterization presented lower values than the vinasse, except for potassium, sulfide and ammonia nitrogen. On the other hand, primers used to amplify the 16S-rDNA genes for the domains Archaea and Bacteria showed the presence of microorganisms which favor methane production at the optimum organic loading rate.

Conclusions

The modified UASB reactor proposed in this study provided a successful treatment of the vinasse obtained from hydrous ethanol production.Methanogen groups (Methanobacteriales and Methanosarcinales) detected by PCR during operational optimum OLR of the modified UASB reactor, favored methane production.

     

Brachy-syndactyly caused by loss of Sfrp2 function

Wnt signaling pathways are regulated both at the intracellular and extracellular levels. During embryogenesis, the in vivo effects of the secreted frizzled-related protein (Sfrp) family of Wnt inhibitors are poorly understood. Here, we show that inactivation of Sfrp2 results in subtle limb defects in mice with mesomelic shortening and consistent shortening of all autopodal elements that is clinically manifested as brachydactyly. In addition, there is soft-tissue syndactyly of the hindlimb. The brachydactyly is caused by decreased chondrocyte proliferation and delayed differentiation in distal limb chondrogenic elements. These data suggest that Sfrp2 can regulate both chondrogenesis and regression of interdigital mesenchyme in distal limb. Sfrp2 can also repress canonical Wnt signaling by Wnt1, Wnt9a, and Wnt4 in vitro. Sfrp2-/- and TOPGAL/Sfrp2-/- mice have a mild increase in beta-catenin and beta-galactosidase staining, respectively, in some phalangeal elements. This however does not exclude a potential concurrent effect on non-canonical Wnt signaling in the growth plate. In combination with what is known about BMP and Wnt signaling in human brachydactylies, our data establish a critical role for Sfrp2 in proper distal limb formation and suggest SFPR2 could be a novel candidate gene for human brachy-syndactyly defects.     

Insulin-stimulated ribosomal protein synthesis in maize embryonic axes during germination

Addition of insulin to maize seed ( Zea mays L. cv. Chalqueño) was found to accelerate germination and seedling growth. Insulin-stimulated maize axes showed enhancement of 35 S-methionine incorporation into ribosomal proteins (rp) and mobilization of S₆ rp mRNA into polysomes. Increase in S₆ rp phosphorylation of the small ribosomal subunit (40S) was observed in 32 P-orthophosphate pulse-labeled experiments when maize axes were stimulated by insulin. Application of either wortmannin or rapamycin, inhibitors of protein kinases of the insulin transduction pathway, abolished the insulin stimulatory effect on S₆ rp phosphorylation and on ribosomal protein synthesis. The above data are interpreted as an indication of the existence of an insulin-stimulated signal transduction pathway in maize tissues that is involved in the regulation of translation.     

In vitro analysis of the melanoma/endothelium interaction increasing the release of soluble intercellular adhesion molecule 1 by endothelial cells

Melanoma cells constitutively release intercellular adhesion molecule 1 (ICAM-1) as soluble ICAM-1 (sICAM-1), and its levels are elevated in melanoma patients and correlate with disease progression. However, this correlation is not absolute, suggesting that specific characteristics of neoplastic cells and/or ICAM-1-positive non-neoplastic cells may influence the amounts of circulating sICAM-1. In this study, we found a weak correlation (r = 0.55; r ² = 0.3) between sICAM-1 release by 40 metastatic melanomas (36 primary cultures and 4 cell lines), and ICAM-1 expression on neoplastic cells. In addition, melanoma-secreted interleukin-1α (IL-1α) (1/40) but not vascular endothelial growth factor (VEGF) (29/40), significantly (P < 0.05) up-regulated the shedding of sICAM-1 by human umbilical vein endothelial cells (HUVEC). This was completely abolished by IL-1α/β neutralizing antibodies both at the protein and mRNA level. Altogether, our results suggest that (i) the extent of sICAM-1 release is distinctive for individual melanomas and can be independent of ICAM-1 expression; (ii) tumor endothelia may sustain levels of sICAM-1 in selected melanomas; (iii) melanoma-released VEGF does not affect ICAM-1 expression and sICAM-1 release by HUVEC. Melanoma-derived sICAM-1 inhibits cell-mediated cytotoxicity of melanoma cells; therefore, constitutive levels of sICAM-1 release and IL-1α secretion by individual melanomas can differentially influence tumor progression and the clinical effectiveness of cytotoxic-cell-based vaccines. Received: 15 October 1998 / Accepted: 17 February 1999     

Shedding of the 67-kD laminin receptor by human cancer cells

The 67-kD laminin receptor (67LR) is a cell membrane-associated molecule exhibiting high affinity for the basement membrane glycoprotein, laminin. While export of the 67LR toward the extracellular matrix has been recently suggested by electron microscopy studies, there is to date no evidence of shedding of the 67LR from cells. Using two monoclonal antibodies directed against the 67LR, we developed a double-determinant radioimmunoassay that demonstrates that the 67LR is released from cancer cells into the culture medium. The shed molecule exhibited the same apparent molecular weight as that of the membrane-associated 67LR, suggesting that no proteolytic cleavage is involved in the process. Furthermore, we demonstrate that the 67LR is not anchored to the membrane through a glycolsyl-phosphatidylinositol bridge. However, the observation that lactose increased the release of 67LR suggests that a lectin-type interaction is involved in the cell membrane association of this laminin binding protein and the cell surface. Interestingly, the released 67LR recovered after HPLC gel filtration was found free as well as associated to high molecular weight complexes. The free 67LR retained its ability to bind to the cell surface. Our study is the first demonstration that the 67LR is effectively shed by cancer cells. The released free 67LR could play an important role in modulating interactions between cancer cells and laminin during tumor invasion and metastasis. © 1996 Wiley-Liss, Inc.     

Hemorrhagic, coagulant and fibrino(geno)lytic activities of crude venom and fractions from mapanare (Bothrops colombiensis) snakes

Bothrops colombiensis venom from two similar geographical locations were tested for their hemostatic functions and characterized by gel-filtration chromatography and SDS-PAGE electrophoresis. The snakes were from Caucagua and El Guapo towns of the Venezuelan state of Miranda. Fibrino(geno)lytic, procoagulant, hemorrhagic, lethal activities, gel-filtration chromatography and SDS-PAGE profiles were analyzed and compared for both venoms. The highest hemorrhagic activity of 5.3 mug was seen in El Guapo venom while Caucagua venom had the lowest LD(50) of 5.8 mg/kg. Both venoms presented similar thrombin-like activity. El Guapo showed a factor Xa-like activity two times higher than Caucagua. Differences were observed in kallikrein-like and t-PA activities, being highest in El Guapo. Caucagua venom showed the maximum fibrin lysis. Both crude venom runs on Sephadex G-100 chromatography gave fraction SII with the high fibrinolytic activity. Proteases presented in SII fractions and eluted from Benzamidine-Sepharose (not bound to the column) provoked a fast degradation of fibrinogen alpha chains and a slower degradation of beta chains, which could possibly be due to a higher content of alpha fibrinogenases in these venoms. The fibrinogenolytic activity was decreased by metalloprotease inhibitors. The results suggested that metalloproteases in SII fractions were responsible for the fibrinolytic activity. The analysis of samples for fibrin-zymography of SII fractions showed an active band with a molecular mass of approximately 30 kDa. These results reiterate the importance of using pools of venoms for antivenom immunization, to facilitate the neutralization of the maximum potential number of toxins.