In vitro and in vivo treatment of colon cancer by VIP antagonists

Vasoactive intestinal peptide (VIP) is secreted from many cancer lines and VIP binding was observed in many tumors. We have shown before that VIP antagonists are potent inhibitors of neoplastic growth of neuroblastoma, lung and breast cancer cells in vitro. Here, the cultured colon cancer cell line HCT-15 that exhibited VIP receptor expression was treated with the VIP hybrid antagonist neurotensin(6-11)VIP(7-28). The antineoplastic activity was assessed by thymidine incorporation. Neurotensin(6-11)VIP(7-28) efficiently inhibited cancer growth with a maximal effect at nanomolar concentrations. Once the inhibitory properties of the VIP antagonist on colon cancer cells were established, the in vivo curative effects were analyzed. Sprague-Dawley rats were injected with azoxymethane (AOM) (15 mg/kg/week) for 2 weeks, providing artificial induction of colon tumors. The rats were then allocated into four experimental groups: (1) receiving no treatment; (2) receiving treatment with saline; (3, 4) receiving treatment with 10 or 20 microg of neurotensin(6-11)VIP(7-28), respectively. After 10 weeks of daily injections, rats were sacrificed and tumors assessed for stage, volume, location, differentiation and lymphocytic infiltrate. Embedded mucosa was assessed for dysplastic crypts. Results showed that the antagonist treatment reduced the tumor volume, staging, lymphocyte infiltrate and the number of dysplastic crypts. Thus, neurotensin(6-11)VIP(7-28) could serve as an effective cancer treatment and a preventing agent.     

A heterogeneous tRNA granule structure exhibiting rapid,bi-directional neuritic transport

mRNA translation is regulated by diverse mechanisms that converge at the initiation and elongation steps to determine the rate, profile, and localization of proteins synthesized. A consistently relevant feature of these mechanisms is the spatial re-distribution of translation machinery, a process of particular importance in neural cells. This process has, however, been largely overlooked with respect to its potential role in regulating the local concentration of cytoplasmic tRNAs, even as a multitude of data suggest that spatial regulation of the tRNA pool may help explain the remarkably high rates of peptide elongation. Here, we report that Cy3/Cy5-labeled bulk tRNAs transfected into neural cells distribute into granule-like structures – “tRNA granules” – that exhibit dynamic mixing of tRNAs between granules and rapid, bi-directional vectorial movement within neurites. Imaging of endogenous tRNA_gly and tRNA_lys by fluorescent in situ hybridization revealed a similar granular distribution of tRNAs in somata and neurites; this distribution was highly overlapping with granules imaged by introduction of exogenous Cy5-tRNA_thr and Cy3-tRNA_val. A subset of tRNA granules located in the cell body, neurite branch points and growth cones displayed fluorescence resonance energy transfer (FRET) between Cy3 and Cy5-labeled tRNAs indicative of translation, and co-localization with elongation machinery. A population of smaller, rapidly trafficked granules in neurites lacked FRET and showed poor colocalization with translation initiation and elongation factors, suggesting that they are a translationally inactive tRNA transport particle. Our data suggest that tRNAs are packaged into granules that are rapidly transported to loci where translation is needed, where they may greatly increase the local concentration of tRNAs in support of efficient elongation. The potential implications of this newly described structure for channeling of elongation, local translation, and diseases associated with altered tRNA levels or function are discussed.     

4‐phenylbutyric acid—Identity crisis; can it act as a translation inhibitor?

Loss of proteostasis can occur due to mutations, the formation of aggregates, or general deficiency in the correct translation and folding of proteins. These phenomena are commonly observed in pathologies, but most significantly, loss of proteostasis characterizes aging. This loss leads to the chronic activation of stress responses and has a generally deleterious impact on the organism. While finding molecules that can alleviate these symptoms is an important step toward solutions for these conditions, some molecules might be mischaracterized on the way. 4‐phenylbutyric acid (4PBA) is known for its role as a chemical chaperone that helps alleviate endoplasmic reticulum (ER) stress, yet a scan of the literature reveals that no biochemical or molecular experiments have shown any protein refolding capacity. Here, we show that 4PBA is a conserved weak inhibitor of mRNA translation, both in vitro and in cellular systems, and furthermore—it does not promote protein folding nor prevents aggregation. 4PBA possibly alleviates proteostatic or ER stress by inhibiting protein synthesis, allowing the cells to cope with misfolded proteins by reducing the protein load. Better understanding of 4PBA biochemical mechanisms will improve its usage in basic science and as a drug in different pathologies, also opening new venues for the treatment of different diseases.     

Comparison of movement and metabolism of indole-3-acetic acid and indole-3-butyric acid in mung bean cuttings

Indole-3-butyric acid (IBA) was much more effective than indole-3-acetic acid (IAA) in inducing adventitious root formation in mung bean ( Vigna radiata L.) cuttings. Prolonging the duration of treatment with both auxins from 24 to 96 h significantly increased the number of roots formed. Labelled IAA and IBA applied to the basal cut surface of the cuttings were transported acropetally. With both auxins, most radioactivity was detected in the hypocotyl, where roots were formed, but relatively more IBA was found in the upper sections of the cuttings. The rate of metabolism of IAA and IBA in these cuttings was similar. Both auxins were metabolized very rapidly and 24 h after application only a small fraction of the radioactivity corresponded to the free auxins. Hydrolysis with 7 M NaOH indicates that conjugation is the major pathway of IAA and IBA metabolism in mung bean tissues. The major conjugate of IAA was identified tentatively as indole-3-acetylaspartic acid, whereas IBA formed at least two major conjugates. The data indicate that the higher root-promoting activity of IBA was not due to a different transport pattern and/or a different rate of conjugation. It is suggested that the IBA conjugates may be a better source of free auxin than those of IAA and this may explain the higher activity of IBA.     

LTC: a novel algorithm to improve the efficiency of contig assembly for physical mapping in complex genomes

Background  

Physical maps are the substrate of genome sequencing and map-based cloning and their construction relies on the accurate assembly of BAC clones into large contigs that are then anchored to genetic maps with molecular markers. High Information Content Fingerprinting has become the method of choice for large and repetitive genomes such as those of maize, barley, and wheat. However, the high level of repeated DNA present in these genomes requires the application of very stringent criteria to ensure a reliable assembly with the FingerPrinted Contig (FPC) software, which often results in short contig lengths (of 3-5 clones before merging) as well as an unreliable assembly in some difficult regions. Difficulties can originate from a non-linear topological structure of clone overlaps, low power of clone ordering algorithms, and the absence of tools to identify sources of gaps in Minimal Tiling Paths (MTPs).     

Mutations disrupting selenocysteine formation cause progressive cerebello-cerebral atrophy

The essential micronutrient selenium is found in proteins as selenocysteine (Sec), the only genetically encoded amino acid whose biosynthesis occurs on its cognate tRNA in humans. In the final step of selenocysteine formation, the essential enzyme SepSecS catalyzes the conversion of Sep-tRNA to Sec-tRNA. We demonstrate that SepSecS mutations cause autosomal-recessive progressive cerebellocerebral atrophy (PCCA) in Jews of Iraqi and Moroccan ancestry. Both founder mutations, common in these two populations, disrupt the sole route to the biosynthesis of the 21st amino acid, Sec, and thus to the generation of selenoproteins in humans.     

Evidence that oocyte maturation induced by an oncogenic ras-p21 protein and insulin is mediated by overlapping yet distinct mechanisms

We have recently shown that a peptide (residues 35–47) from a functional region of the ras p21 protein, thought to be involved in the binding of p21 to GTPase activating protein, the antibiotic azatyrosine, known to induce the ras-recision gene, and the selective protein kinase C inhibitor, CGP 41 251, all inhibit oncogenic p21 protein-induced maturation of oocytes in a dose-dependent manner. We now show that these three agents only partially inhibit insulin-induced oocyte maturation, known to be dependent on activation of cellular p21 protein. On the other hand, the anti-p21 protein antibody Y13–259 completely inhibits both insulin- and oncogenic p21 protein-induced maturation as does a tetrapeptide, CVIM, known to block the enzyme farnesyl transferase which covalently attaches the farnesyl moiety to the p21 protein allowing it to attach to the cell membrane. Our results suggest that while the oncogenic and insulin-activated normal p21 proteins share certain elements of their signal transduction pathways in common, these pathways diverge and allow for selective inhibition of the oncogenic pathway.     

A systems approach to morphogenesis in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>: I. AGNS database

In systems biology, study of a complex and multicomponent system, such as morphogenesis, comprises accumulation of data on morphogenetic processes in databases, classification and logical analysis of this information, and computer simulation of the processes in question using the data accumulated and the results of their analysis. This paper describes realization of the first steps in a systems study of morphogenesis (annotating research papers, compiling information in a database, data systematization, and their logical analysis) by the example of Arabidopsis thaliana, a model object in plant molecular biology. The database AGNS (Arabidopsis GeneNet Supplementary; http://wwwmgs.bionet.nsc.ru/agns) contains the experimentally confirmed information from published papers on specific features of gene expression and phenotypes of wild-type, mutant, and transgenic A. thaliana plants. AGNS queries and logical data analysis with the aid of specially developed software makes it possible to model various morphogenetic processes from gene expression to functioning of gene networks and their contribution to the development of certain traits.     

Natural β-carotene and whole body irradiation in rats

β-Carotene and other carotenoids are reported to be potent free radical quenchers, singlet oxygen scavengers, and lipid antioxidants. Whole-body irradiation is known to cause an immunosuppression effect in mammals through the possible initiation and production of reactive oxygen species. We decided to test the possible antioxidative effect against whole-body irradiation of a natural β-carotene, composed of equal amounts of the all-trans and 9-cis isomers, obtained from the unicellular alga Dunaliella bardawil. Rats were fed on ground commercial food enriched with natural β-carotene (50 mg/kg diet). On completion of 1 week with β-carotene, the rats were exposed to a single dose of 4 Gy whole-body irradiation, after which their livers and blood were removed for β-carotene and retinol analysis in comparison with control livers of animals irradiated or not, or supplemented with β-carotene after irradiation. A normal increase in body weight with no ill effects was noted in the groups of rats whose diet was supplemented by β-carotene before and after irradiation, compared with the reduction in the specific growth rate in the group of rats irradiated without β-carotene. Liver β-carotene and retinol decreased significantly after irradiation compared with the rats which were not irradiated. This decrease was not shown in rats fed β-carotene prior to irradiation, and the effect of irradiation was partially cured by supplementation with β-carotene after irradiation. High-pressure liquid chromatography (HPLC) analysis of the irradiated animals showed a selective decline in 9-cis β-carotene and in retinol over all-trans β-carotene and retinyl esters. These results suggest that 9-cis β-carotene and retinol protect in vivo against the cellular damage by free radicals induced after whole-body irradiation. Received: 14 May 1996 / Accepted in revised form: 24 June 1996