E2F and Ras synergize in transcriptionally activating p14ARF expression

The INK4a/ARF locus, which is frequently inactivated in human tumors, encodes two distinct tumor suppressive proteins, ARF and p16INK4a. ARF stabilizes and activates p53 by negating the effects of mdm2 on p53. Furthermore, its function is not restricted to the p53 pathway and it also inhibits cell proliferation in cells lacking p53. Expression of ARF is up-regulated in response to a number of oncogenic stimuli including E2F1. We show here that while oncogenic Ras does not significantly affect p1(4AR)F expression in normal human cells it activates p1(4AR)F in cells containing deregulated E2F. Moreover, oncogenic Ras and E2F1 synergize in activating p1(4AR)F expression. Activation of p1(4AR)F promoter by E2F1 persists in the absence of the consensus E2F-binding sites in this promoter, indicating that this activation also occurs through non- canonical binding sites. The activation by oncogenic Ras requires both E2F and Sp-1 activity, demonstrating the complex regulation of p14(ARF) in response to oncogenic stimuli.     

Substrate range of acetohydroxy acid synthase I from Escherichia coli in the stereoselective synthesis of alpha-hydroxy ketones

Acetohydroxy acid synthase I appears to be the most effective of the AHAS isozymes found in Escherichia coli in the chiral synthesis of phenylacetyl carbinol from pyruvate and benzaldehyde. We report here the exploration of a range of aldehydes as substrates for AHAS I and demonstrate that the enzyme can accept a wide variety of substituted benzaldehydes, as well as heterocyclic and heteroatomic aromatic aldehydes, to produce chiral carbinols. The active site of AHAS I does not appear to impose serious steric constraints on the acceptor substrate. The influence of electronic effects on the reaction has been probed using substituted benzaldehydes as substrates. The electrophilicity of the aldehyde acceptor substrates is most important to their reactivity, but the lipophilicity of substituents also affects their reactivity. AHAS I is an effective biosynthetic platform for production of a variety of alpha-hydroxy ketones, compounds with considerable potential as pharmacological precursors.     

Role of a conserved arginine in the mechanism of acetohydroxyacid synthase: catalysis of condensation with a specific ketoacid substrate

The thiamin diphosphate (ThDP)-dependent bio-synthetic enzyme acetohydroxyacid synthase (AHAS) catalyzes decarboxylation of pyruvate and specific condensation of the resulting ThDP-bound two-carbon intermediate, hydroxyethyl-ThDP anion/enamine (HEThDP(-)), with a second ketoacid, to form acetolactate or acetohydroxybutyrate. Whereas the mechanism of formation of HEThDP(-) from pyruvate is well understood, the role of the enzyme in control of the carboligation reaction of HEThDP(-) is not. Recent crystal structures of yeast AHAS from Duggleby's laboratory suggested that an arginine residue might interact with the second ketoacid substrate. Mutagenesis of this completely conserved residue in Escherichia coli AHAS isozyme II (Arg(276)) confirms that it is required for rapid and specific reaction of the second ketoacid. In the mutant proteins, the normally rapid second phase of the reaction becomes rate-determining. A competing alternative nonnatural but stereospecific reaction of bound HEThDP(-) with benzaldehyde to form phenylacetylcarbinol (Engel, S., Vyazmensky, M., Geresh, S., Barak, Z., and Chipman, D. M. (2003) Biotechnol. Bioeng. 84, 833-840) provides a new tool for studying the fate of HEThDP(-) in AHAS, since the formation of the new product has a very different dependence on active site modifications than does acetohydroxyacid acid formation. The effects of mutagenesis of four different residues in the site on the rates and specificities of the normal and unnatural reactions support a critical role for Arg(276) in the stabilization of the transition states for ligation of the incoming second ketoacid with HEThDP(-) and/or for the breaking of the product-ThDP bond. This information makes it possible to engineer the active site so that it efficiently and preferentially catalyzes a new reaction.     

Adaptation of plants to altered shoot orientation relative to the gravity vector

Wheat Triticum aestivum L., carrots Daucus carota L., Chinese cabbage Brassica pekinensis Rupr., and African marigold Tagetes patula L. were grown at natural and inverted orientation in the Earth gravitational field. Light vector was set unidirectional or opposite directional relative to the gravity vector. Shoot orientation relative to the gravity vector was set natural or invert. Plants grew in the special pots furnished with plane or cylindrical hydrophilic porous membranes. The membrane allowed to stabilize a water potential in the root zone at the fixed level. Seeds were put into a fiber ion-exchange artificial soil overlaying horizontal hydrophilic plates of porous titanium or anchored to porous metal-ceramic tubes. Plants grew at the PPF level 550 +/- 20 micromoles/(m2 s) during 24-hr lighting and at the water potential level at the membrane surface (-1.00) +/- 0.08 kPa. Normal plants were obtained both at the natural and at the inverse shoot orientation in the all experiments. The wheat plants were yielded healthy germinating seeds no matter plant orientation. In the inverse orientation, no negative influence for plant biomass accruing was marked, but the increasing of shoot to root mass ratio was considerable. However carrot root crop mass decreasing was not revealed in the inverse orientation. The results demonstrated substantial dependence of morphological and physiological characteristics of higher plants on the gravity factor.     

Association between Tumorigenic Potential and the Fate of Cancer Cells in a Syngeneic Melanoma Model

The self-renewal potential of a cancer cell can be estimated by using particular assays, which include xenotransplantation in immunocompromised animals or culturing in non-adherent serum-free stem-cells media (SCM). However, whether cells with self-renewal potential actually contribute to disease is unknown. Here we investigated the tumorigenic potential and fate of cancer cells in an in-vivo melanoma model. We examined cell lines which were derived from the same parental line: a non-metastatic cell line (K1735/16), a metastatic cell line (K1735/M4) and a cell line which was selected in non-adherent conditions (K1735/16S). All cell lines exhibited similar proliferation kinetics when grown on culture plates. K1735/16 cells grown in soft agar or in suspension non-adherent conditions failed to form colonies or spheroids, whereas the other cell lines showed prominent colonogenicity and spheroid formation capacity. By using sphere limiting dilution analysis (SLDA) in serum-free media, K1735/16S and K1735/M4 cells grown in suspension were capable of forming spheroids even in low frequencies of concentrations, as opposed to K1735/16 cells. The tumorigenic potential of the cell lines was determined in SCID mice using intra footpad injections. Palpable tumors were evident in all mice. In agreement with the in-vitro studies, the K1735/M4 cell line exhibited the highest growth kinetics, followed by the K1735/16S cell line, whereas the K1735/16 cell line had the lowest tumor growth potential (P<0.001). In contrast, when we repeated the experiments in syngeneic C3H/HeN mice, the K1735/16 cell line produced macroscopic tumors 30–100 days after injection, whereas K1735/M4 and K1735/16S derived tumors regressed spontaneously in 90–100% of mice. TUNEL analysis revealed significantly higher number of apoptotic cells in K1735/16S and K1735/M4 cell line-derived tumors compared to K1735/16 tumors (P<0.001). The models we have examined here raised the possibility, that cells with high-tumorigenic activity may be more immunogenic and hence are more susceptible to immune-regulation.     

Biofilm formation onto starch fibres by Bacillus subtilis governs its successful adaptation to chickpea milk

Beneficial biofilms may confer effective adaptation to food matrices that assist bacteria in enduring hostile environmental conditions. The matrices, for instance, dietary fibres of various food products, might serve as a natural scaffold for bacterial cells to adhere and grow as biofilms. Here, we report on a unique interaction of Bacillus subtilis cells with the resistant starch fibresof chickpea milk (CPM), herein CPM fibres, along with the production of a reddish-pink pigment. Genetic analysis identified the pigment as pulcherrimin, and also revealed the involvement of Spo0A/SinI pathway in modulating the observed phenotypes. Besides, through successful colonization of the CPM fibres, the wild-type cells of B. subtilis displayed enhanced survivability and resilience to environmental stress, such as heat and in vitro gastrointestinal treatments. In total, we infer that the biofilm formation on CPM fibres is an adaptation response of B. subtilis for strategic survival.