New exopolysaccharides produced by Aureobasidium pullulans grown on glucosamine

The polysaccharides produced by Aureobasidium pullulans, grown using glucosamine as the carbon source, were investigated by means of methylation analysis, affinity chromatography and NMR spectroscopy. The results indicated that, besides a small amount of pullulan, this micro-organism was capable of producing-in low yields-mixtures of at least two different complex polysaccharides containing mainly mannose and galactose. (1)H NMR spectra of two fractions obtained by lectin affinity chromatography indicated that one polymer was constituted exclusively of mannose residues while the other contained both galactofuranosyl and mannopyranosyl residues.     

Embryo recovery and pregnancy rates after the delay of ovulation and fixed time insemination in superstimulated beef cows

The aim of this study was to evaluate the effect of delaying ovulation subsequent to superstimulation of follicular growth in beef cows (Bos indicus) on embryo recovery rates and the capacity of embryos to establish pregnancies. Ovulation was delayed by three treatments using either progesterone (CIDR-B) or a GnRH agonist (deslorelin). Multiparous Nelore cows (n = 24) received three of four superstimulation treatments in an incomplete block design (n = 18 per group). Cows in Groups CTRL, P48 and P60 were treated with a CIDR-B device plus estradiol benzoate (EB, 4 mg, i.m.) on Day-5, while cows in Group D60 were implanted with deslorelin on Day-7. Cows were superstimulated with FSH (Folltropin-V, 200 mg), from Day 0 to 3, using twice daily injections in decreasing amounts. All cows were treated with a luteolytic dose of prostaglandin on Day 2 (08:00 h). CIDR-B devices were removed as follows: Group CTRL, Day 2 (20:00 h); Group P48, Day 4 (08:00 h); Group P60, Day 4 (20:00 h). Cows in Group CTRL were inseminated at 10, 20 and 30 h after first detected estrus. Ovulation was induced for cows in Group P48 (Day 4, 08:00 h) and Groups P60 and D60 (Day 4, 20:00 h) by injection of LH (Lutropin, 25 mg, i.m.), and these cows were inseminated 10 and 20 h after treatment with LH. Embryos were recovered on Days 11 or 12, graded and transferred to synchronized recipients. Pregnancies were determined by ultrasonography around Day 100. Data were analyzed by mixed procedure, Kruskal-Wallis and Chi-square tests. The number of ova/embryos, transferable embryos (mean +/- SEM) and pregnancy rates (%) were as follows, respectively: Group CTRL (10.8+/-1.8, 6.1+/-1.3, 51.5), P48 (12.6+/-1.9, 7.1+/-1.0, 52.3), P60 (10.5+/-1.6, 5.7+/-1.3, 40.0) and D60 (10.3+/-1.7, 5.0+/-1.2, 50.0). There were no significant differences among the groups (P > 0.05). It was concluded that fixed time AI in association with induced ovulation did not influence embryo recovery. Furthermore, pregnancy rates in embryos recovered from cows with delayed ovulation were similar to those in embryos obtained from cows treated with a conventional superstimulation protocol.     

Chromatographic determination of 7,8-methylenedioxy-4H-2,3-benzodiazepin-4-ones in rat plasma: relationship to their anticonvulsant activity

The present investigation was designed to develop an assay suitable for pharmacokinetic studies of new compounds, i.e. the novel 7,8-methylenedioxy-4H-2,3-benzodiazepin-4-one derivatives (2a and 2b), acting as non-competitive AMPA-receptor antagonists. A reversed-phase high-performance liquid chromatographic method has been developed to determine the time-course of plasma concentrations of derivatives 2a and 2b administered intraperitoneally to Sprague-Dawley rats. The separation of compounds studied and a N-methyl-2,3-benzodiazepin-4-one derivative as internal standard (I.S.) from plasma, were carried out by liquid-liquid extraction using diethyl ether. The samples were injected onto the analytical column (Partisil 10 ODS) eluted with acetonitrile/0.01 M acetate buffer (pH 5.3) at a flow-rate of 2 ml/min and detected at 240 nm. Compounds 2a, 2b and I.S. gave retention times of 8.5, 5.25 and 11.1 min, respectively. The selectivity of the method was satisfactory. The mean recovery from spiked rat plasma ranged from 86.7 to 91.6% for 2a, and from 85.1 to 87.0% for 2b. The procedures were validated with a good reproducibility and linear response from 0.0625 to 2 microg/ml, with a regression coefficient of 0.9932 for 2a and 0.9854 for 2b. The lower limit of quantification (LOQ) was taken as 15 ng/ml for the two compounds. 2a and 2b showed no signs of significant degradation in rat plasma during storage at -20 degrees C and following freeze/thaw cycles. Moreover, plasma levels of the tested compounds have been correlated with their anticonvulsant activity, determined in vivo in genetically epilepsy-prone rats. Due to its sensitivity, the method was suitable for application to pharmacokinetic study.     

Activation of Notch-1 signaling maintains the neoplastic phenotype in human Ras-transformed cells

Truncated Notch receptors have transforming activity in vitro and in vivo. However, the role of wild-type Notch signaling in neoplastic transformation remains unclear. Ras signaling is deregulated in a large fraction of human malignancies and is a major target for the development of novel cancer treatments. We show that oncogenic Ras activates Notch signaling and that wild-type Notch-1 is necessary to maintain the neoplastic phenotype in Ras-transformed human cells in vitro and in vivo. Oncogenic Ras increases levels and activity of the intracellular form of wild-type Notch-1, and upregulates Notch ligand Delta-1 and also presenilin-1, a protein involved in Notch processing, through a p38-mediated pathway. These observations place Notch signaling among key downstream effectors of oncogenic Ras and suggest that it might be a novel therapeutic target.     

Increase in ceramide level alters the lysosomal targeting of cathepsin D prior to onset of apoptosis in HT-29 colon cancer cells

Ceramide has been suggested as an important mediator of apoptosis. In HT-29 colorectal cancer cells increased ceramide levels, induced by exogenous N-acetylsphingosine (NAS, also known as C2-ceramide) or by 1-phenyl-2-(decanoylamino)-3-morpholino-1-propanol (PDMP), inhibited the transport and processing of cathepsin D (CD), a lysosomal protease implicated in apoptosis of tumour cells. C2-dihydroceramide (DH-C2), an inactive analogue of NAS, had no effect on CD transport and maturation. The treatment with either NAS or PDMP was revealed to be cytotoxic for HT-29 cells and led to cell death with classical features of apoptosis. Morphological signs of apoptosis and DNA fragmentation became apparent only between 24 and 48 h of incubation and poly(ADP ribose)-polymerase cleavage, a hallmark of caspase 3 activity, occurred no earlier than 8 h from incubation. Secretion of proCD was almost abolished and the formation of double-chain mature CD was reduced and delayed by NAS, whereas PDMP largely inhibited the lysosomal targeting and maturation of proCD. NAS- and PDMP-induced alteration of proCD transport and maturation were apparent already 2 h after incubation with the drugs, which is much earlier than when classical biochemical and morphological evidence of apoptosis could be detected. These data indicate that alteration of CD (and possibly of other glycoproteins) transport along the secretory pathway due to increased levels of cell-associated ceramide is an early event in cells undergoing apoptosis.     

Effects of microgravity and hypergravity on early development stages of Xeonopus laevis

The aim of this work is to evaluate the development of X.l. in modified gravity conditions. The simulation of hyper and microgravity was performed utilizing: an hyperfuge, a Clinostat and later on a Random Positioning Machine (RPM, 3d Clinostat). The effect of hypergravity on embryos is significantly higher than that of microgravity; the exposure of embryos to 3xg for 3 days before and after hatch causes an activation of HSP-60 and HSP-70. Embryos exposed to 3xg during the first 3 days of development are very sensitive and show a retard of development, with a lower content of DNA, neutral glycolipids and gangliosides compared to controls.     

The Guanine Nucleotide Exchange Factor ARNO mediates the activation of ARF and phospholipase D by insulin

Background  

Phospholipase D (PLD) is involved in many signaling pathways. In most systems, the activity of PLD is primarily regulated by the members of the ADP-Ribosylation Factor (ARF) family of GTPases, but the mechanism of activation of PLD and ARF by extracellular signals has not been fully established. Here we tested the hypothesis that ARF-guanine nucleotide exchange factors (ARF-GEFs) of the cytohesin/ARNO family mediate the activation of ARF and PLD by insulin.     

Collagen fibrils are differently organized in weight-bearing and not-weight-bearing regions of pig articular cartilage

The magnetic resonance (MR) appearance of the weight-bearing ("loaded") and not-weight-bearing ("unloaded") regions in T(2)-weighted images of pig articular cartilage is different. On the hypothesis that this difference may be ascribed, at least in part, to a different collagen fibre organization in the two regions, this organization was studied using biochemical, histological, and X-ray diffraction methods. While the mean concentrations of collagen and of its cross-links were the same in the two regions, a regular small angle X-ray diffraction pattern was observed only for the habitually "loaded" tissue. It was also seen by light microscopy that the four typical functional zones were well displayed in the "loaded" cartilage whereas they were not clearly depicted in the "unloaded" tissue. Collagen presented a high concentration of fibrils forming an intricate and dense meshwork at the surface of both "loaded" and "unloaded" cartilage. A second zone of high collagen concentration was present at the upper layer of the deep zone of "loaded" cartilage. By contrast, this lamina of highly concentrated fibrils was lacking in "unloaded" cartilage and collagen fibrils appear thinner. Our study proves that the organization of collagen fibres is different for the "loaded" and "unloaded" regions of articular cartilage. It also suggests that this different organization may influence the MR appearance of the tissue. J. Exp. Zool. 287:346-352, 2000.     

Behaviour of interleukin-2 serum levels in advanced non-small-cell lung cancer patients: relationship with response to therapy and survival

 Interleukin(IL)-2 is a T helper (Th) 1 type cytokine that has been shown to play an important role in antitumour immune responses. In this study, the prognostic significance of serum IL-2 levels was investigated in 60 advanced non-small-cell lung cancer (NSCLC) patients. IL-2 serum levels were determined before chemotherapy, at the end of chemotherapy and during follow-up, using a commercially available enzyme-linked immunoadsorbent assay kit. The results were analysed according to the response to therapy and were used to generate a model predicting overall survival and time to treatment failure. All 60 patients were shown to have higher IL-2 serum levels than controls (P < 0.0001). Stage IV patients had significantly lower IL-2 levels than stage III patients (P < 0.0001), although they were still significantly higher than controls (P < 0.0001). It is interesting that, when patients were divided into responders and non-responders according to the response to therapy, the former were shown to have significantly higher pre-chemotherapy levels than the latter (P < 0.0001). Moreover, a further significant increase in IL-2 serum levels (P=0.004) and a significant decrease (P < 0.0001) were shown in responders and non-responders, respectively at the end of the therapy. Using univariate and multivariate analyses, both overall survival and time to treatment failure were shown to be affected by the mean pathological levels of IL-2. Furthermore, the prognostic significance of the serum level of IL-2 was confirmed by the stepwise regression analysis. In conclusion, determination of pre-treatment IL-2 serum levels was shown to be of independent prognostic utility in patients with advanced NSCLC; therefore, its possible use for prediction of outcome is proposed. Received: 16 March 2000 / Accepted: 27 July 2000     

Microhabitat and morphometric variation of two Chaetophoracean (Chaetophorales, Chlorophyta) species in tropical streams of southeastern Brazil

Two populations of Chaetophora elegans (Roth) C. Agardh and two of Stigeoclonium helveticum Vischer were investigated for microhabitat characteristics and morphological variation in streams of São Paulo State, southeastern Brazil. Different patterns of microhabitat distribution were found between species investigated. Populations of C, elegans were distributed under relatively narrow microhabitat conditions (high irradiance, low depth, moderate to high current velocity, rocky substrata and lower values of niche width) and showing little morphometric variation (colony diameter, main axis cell size, and apical branch number), Stigeoclonium helveticum occurred under more diverse microhabitat conditions, revealed by lack of significant difference between sampling units with and without the alga and wider niche width, but also exhibited relatively narrow morphometric variation (plant length, main axis cell and ateral branch cell sizes). The narrow microhabitat conditions and smaller niche width of C. elegans can explain its low abundance (percentage cover) in streams from the area studied as well as in other regions of São Paulo State, In contrast, the wider variation of microhabitat conditions and the higher niche widths of S. helveticum suggest that this green alga is able to grow in a high number of stream ecosystems in the region investigated, ranging from undisturbed to highly disturbed habitats. Thus. the results suggest that S. helveticum is a generalist species.