Soluble minerals in chemical evolution

The adsorption of 5-AMP onto solid CaSO₄ · 2H₂O was studied in a saturated suspension as a function of pH and electrolyte concentration. The adsorption is pH-dependent and is directly correlated with the charge on the 5-AMP molecule which is determined by the state of protonation of the N-1 nitrogen of the purine ring and the phosphate oxygens. It is proposed that the binding that occurs between the nucleotide and the salt is electrostatic in nature. The adsorption decreases with increasing ionic strength of the solution which means that in a fluctuating environment of wetting and drying cycles, a biomolecule similar to 5-AMP could be expected to desorb during the drying phase. The results indicate that CaSO₄ · 2H₂O can serve as a concentrating surface for biomolecules. The significance of this is discussed with regard to the possible role of soluble minerals and their surfaces in a geochemical model consistent with the evolution of the Earth and the origin of life.     

Cell-binding domain of endothelial cell thrombospondin: localization to the 70-kDa core fragment and determination of binding characteristics.

Endothelial and other cell types synthesize thrombospondin (TSP), secrete it into their culture medium, and incorporate it into their extracellular matrix. TSP is a large multifunctional protein capable of specific interactions with other matrix components, as well as with cell surfaces, and can modulate cell adhesion to the extracellular matrix. With the aim of understanding the mechanism by which TSP exerts its effect on cell adhesion, we studied the interaction of endothelial cell TSP (EC-TSP) with three different cell types: endothelial cells, granulosa cells, and myoblasts. We find that endothelial cells specifically bind radiolabeled EC-TSP with a Kd of 25 nM, and the number of binding sites is 2.6 X 10(6)/cell. Binding is not inhibitable by the cell-adhesion peptide GRGDS, indicating that the cell-binding site of EC-TSP is not in the RGD-containing domain. Localization of the cell-binding site was achieved by testing two chymotryptic fragments representing different regions of the TSP molecule, the 70-kDa core fragment and the 27-kDa N-terminal fragment, for their ability to bind to the cells. Cell-binding capacity was demonstrated by the 70-kDa fragment but not by the 27-kDa fragment. Binding of both intact [125I]EC-TSP and of the 125I-labeled 70-kDa fragment was inhibited by unlabeled TSP, heparin, fibronectin (FN), monoclonal anti-TSP antibody directed against the 70-kDa fragment (B7-3), and by full serum, but not by heparin-absorbed serum or the cell-adhesion peptide GRGDS. The 70-kDa fragment binds to endothelial cells with a Kd of 47 nM, and the number of binding sites is 5.0 x 10(6)/cell.(ABSTRACT TRUNCATED AT 250 WORDS)     

An integrated genetic linkage map of avocado

 An avocado genomic library was screened with various microsatellite repeats. (A/T)_n and (TC/AG)_n sequences were found to be the most frequent repeats. One hundred and seventy-two positive clones were sequenced successfully of which 113 were found to contain simple sequence repeats (SSR). Polymerase chain reaction primers were designed to the regions flanking the SSR in 62 clones. A GenBank search of avocado DNA sequences revealed 1 sequence containing a (CT)₁₀ repeat. A total of 92 avocado-specific SSR markers were screened for polymorphism using 50 offspring of a cross between the avocado cultivars ‘Pinkerton’ and ‘Ettinger’. Both are standard avocado cultivars which are normally outcrossed and highly heterozygous. Fifty polymorphic SSR loci, 17 random amplified polymorphic DNA (RAPD) and 23 minisatellite DNA Fingerprint (DFP) bands were used to construct the avocado genetic map. The resulting data were analyzed with various mapping programs in order to assess which program best accommodated data from progeny of heterozygous parents. The analyses resulted in 12 linkage groups with 34 markers (25 SSRs, 3 RAPDs and 6 DFP bands) covering 352.6 cM. This initial map can serve as a basis for developing a detailed genomic map and for detection of linkage between markers and quantitative trait loci. Received: 2 April 1996 / Accepted: 28 February 1997     

Prebiotic co-evolution of self-replication and translation or RNA world?

A prebiotic scenario is proposed, based on the recent "domain hypothesis" model (Lahav, 1989, J. molec. Evol. 29, 475-479), suggested for domain propagation of RNA-like molecules in a fluctuating environment. The same system is suggested now not only for the evolution of ribozymes, but also for the evolution of directed peptide synthesis, as follows: Short, self-structured strands (termed prebioectons), each possessing a templatable domain which is chargeable by an amino acid, are the predecessors of tRNA (proto-tRNA). Complementary domains are formed on these prebioectons during an environmental cycle such as wetting-drying, followed by their dissociation from their template domain and ligation, to form the predecessor of mRNA (proto-mRNA). The evolution of directed peptide synthesis is suggested to be based on the ability of the charged prebioectons to attach preferentially to their complementary domains on the proto-mRNA. Two stages of this process are envisioned, namely: (a) Template-directed, random peptide synthesis taking place when non-specifically-charged prebioectons are sequentially attached each to its complementary domain on the proto-mRNA, followed by peptide bond formation. (b) Template-and-sequence-directed peptide synthesis, which can be realized after the "invention" of a catalytic molecule capable of specifically charging a proto-tRNA by an amino acid; this is the crucial evolutionary stage, where a crude genetic code becomes functional. Gradually, catalytic peptides and ribozymes are selected for their functions and evolve, while being encoded in the primitive "memory" of the emerging system. Thus, rather than the RNA monopoly postulated by the RNA World hypothesis, an early co-evolution of primitive enzymes and ribozymes is suggested.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thrombospondin inhibits adhesion of endothelial cells

Adsorption of thrombospondin to a substratum inhibits adhesion of endothelial cells to that substratum. Four hours after plating of cells on glass covered with thrombospondin, the number of cells bound per unit area was only 8% of that bound to fibronectin, and 20% of that which could bind to albumin. While on fibronectin cells assumed a well-spread configuration with time in culture, on thrombospondin they stayed completely round. On surfaces constructed by sequential incubation of glass with thrombospondin and fibronectin or other proteins, thrombospondin retained its inhibitory effect on cell adhesion. Fibronectin surfaces treated with thrombospondin lost 50% of their capacity to adhere endothelial cells. Cell spreading was also greatly impaired. These observations indicate that thrombospondin, which is a component of the extracellular matrix, can modulate adhesion of endothelial cells to the matrix.     

Marker-assisted selection based on a multi-trait economic index in chicken: Experimental results and simulation

A method proposed herein allows simultaneous selection for several production traits, taking into consideration their marginal economic values (i.e. the economic value of a trait's additional unit). This economic index-marker assisted selection (EI-MAS) method is based on the calculation of the predicted economic breeding value (BV), using information on DNA markers that have previously been found to be associated with relevant quantitative trait loci. Based on the proposed method, results with real birds showed that sire progeny performance was significantly correlated with expected performance (r = 0.61-0.76; P = 0.03-0.01). Simulation analysis using a computer program written specifically for this purpose suggested that the relative advantage of EI-MAS would be large for traits with low heritability values. As expected, the response to EI-MAS was higher when the map distance between the marker and the quantitative trait gene was small, and vice versa. A large number of distantly located markers, spread 10 cM apart, yielded higher response to selection than a small number of closely located markers spread 3 cM apart. Additionally, the response to EI-MAS was higher when a large number (ca.150) of progeny was used for the prediction equation.     

Telomere Shortening Sensitizes Cancer Cells to Selected Cytotoxic Agents: In Vitro and In Vivo Studies and Putative Mechanisms

Background

Telomere/telomerase system has been recently recognized as an attractive target for anticancer therapy. Telomerase inhibition results in tumor regression and increased sensitivity to various cytotoxic drugs. However, it has not been fully established yet whether the mediator of these effects is telomerase inhibition per se or telomere shortening resulting from inhibition of telomerase activity. In addition, the characteristics and mechanisms of sensitization to cytotoxic drugs caused by telomerase inhibition has not been elucidated in a systematic manner.

Methodology/Principal Findings

In this study we characterized the relative importance of telomerase inhibition versus telomere shortening in cancer cells. Sensitization of cancer cells to cytotoxic drugs was achieved by telomere shortening in a length dependent manner and not by telomerase inhibition per se. In our system this sensitization was related to the mechanism of action of the cytotoxic drug. In addition, telomere shortening affected also other cancer cell functions such as migration. Telomere shortening induced DNA damage whose repair was impaired after administration of cisplatinum while doxorubicin or vincristine did not affect the DNA repair. These findings were verified also in in vivo mouse model. The putative explanation underlying the phenotype induced by telomere shortening may be related to changes in expression of various microRNAs triggered by telomere shortening.

Conclusions/Significance

To our best knowledge this is the first study characterizing the relative impact of telomerase inhibition and telomere shortening on several aspects of cancer cell phenotype, especially related to sensitivity to cytotoxic drugs and its putative mechanisms. The microRNA changes in cancer cells upon telomere shortening are novel information. These findings may facilitate the development of telomere based approaches in treatment of cancer.     

A thermodynamic analysis of the correlation between active Na+ transport and the rate of oxygen consumption in epithelia

Summary Insulin decreased markedly the adenylyl cyclase activity associated with fat cell membranes purified by centrifugation in sucrose gradients. The hormone effect was not readily evident in crude membrane preparations.The kinetics of this effect indicate that some time was required for the onset of the insulin-induced inactivation. This lag period decreased when the insulin concentration was increased. The hormone dose dependence for adenylyl cyclase inactivation measured at a fixed time (3 min) showed a 10 to 15% decrease in activity at 1 to 30 U per ml insulin; 30 to 40% at 100 to 1000 U per ml; and 75% at 0.1 U per ml.The insulin effect was completely abolished by 0.1mm GMP-P(NH)P, 10mm fluoride, or 50 ng per ml glucagon, or by increasing the Mn⁺⁺ concentration to 4mm. In addition, it was partially reversed by the addition of a fraction from the sucrose gradient, which contained soluble factors.The kinetics of the adenylyl cyclase-catalyzed reaction were studied using ATP or AMP-P(NH)P as adenylyl donor, and Mn⁺⁺ or Mg⁺⁺ as divalent cation, in the absence or presence of insulin. With ATP and Mg⁺⁺ there was a striking reduction of the transient reaction rates after 1.5 min of incubation. Under these conditions the insulin effect was not evident. On the contrary, with ATP and Mn⁺⁺ this spontaneous reduction of activity was less evident; however, in the presence of insulin there was a clear and marked reduction of the transient reaction rate measured after 1.5 min of incubation. With AMP-P(NH)P the kinetic data were qualitatively similar to those observed with ATP.It is concluded that under certain assay conditions adenylyl cyclase may be converted to an inactive enzyme form, and that such a conversion is more evident in the presence of Mg⁺⁺ than with Mn⁺⁺. In the latter case, insulin appears to enhance the rate of this conversion.