Activation of a low specific activity form of DNA polymerase alpha by inositol-1,4-bisphosphate

A low activity form of DNA polymerase alpha immunoaffinity-purified from adult-derived human fibroblasts was activated by interaction with phosphatidylinositol-4-monophosphate, while a high activity form of the enzyme did not interact with phosphatidylinositol-4-monophosphate or its derivatives. Phosphatidylinositol-4-monophosphate was apparently hydrolyzed in the presence of a highly purified low activity form of DNA polymerase alpha, effecting the release of diacylglycerol and the retention of inositol-1,4-bisphosphate by the enzyme complex. The resulting inositol-1,4-bisphosphate/protein complex exhibited increased affinity of binding to DNA template/primer and increased deoxynucleotidyltransferase activity. These data indicate that inositol-1,4-bisphosphate may function as an effector molecule in the activation of a low activity form of human DNA polymerase alpha and suggest that it may function as a second messenger during the initiation of mitosis in stimulated cells.     

Targeted mutagenesis of duplicated genes in soybean with zinc-finger nucleases

We performed targeted mutagenesis of a transgene and nine endogenous soybean (Glycine max) genes using zinc-finger nucleases (ZFNs). A suite of ZFNs were engineered by the recently described context-dependent assembly platform--a rapid, open-source method for generating zinc-finger arrays. Specific ZFNs targeting dicer-like (DCL) genes and other genes involved in RNA silencing were cloned into a vector under an estrogen-inducible promoter. A hairy-root transformation system was employed to investigate the efficiency of ZFN mutagenesis at each target locus. Transgenic roots exhibited somatic mutations localized at the ZFN target sites for seven out of nine targeted genes. We next introduced a ZFN into soybean via whole-plant transformation and generated independent mutations in the paralogous genes DCL4a and DCL4b. The dcl4b mutation showed efficient heritable transmission of the ZFN-induced mutation in the subsequent generation. These findings indicate that ZFN-based mutagenesis provides an efficient method for making mutations in duplicate genes that are otherwise difficult to study due to redundancy. We also developed a publicly accessible Web-based tool to identify sites suitable for engineering context-dependent assembly ZFNs in the soybean genome.     

Heterocyclic ketones as inhibitors of histone deacetylase

Several heterocyclic ketones were investigated as potential inhibitors of histone deacetylase. Nanomolar inhibitors such as 22 and 25 were obtained, the anti-proliferative activity of which were shown to be mediated by HDAC inhibition.     

A web server to locate periodicities in a sequence

Summary : FT is a tool written in C++, which implements the Fourier analysis method to locate periodicities in aminoacid or DNA sequences. It is provided for free public use on a WWW server with a Java interface. Availability : The server address is http://o2.db. uoa.gr/FT Contact : shamodr@atlas.uoa.gr      

Prolongation of Life in Anephric Rats following de novo Renal Organogenesis

One solution to the shortage of human organs available for transplantation envisions growing new organs in situ. This can be accomplished by transplantation of developing organ anlagen/primordia. Allotransplantation of embryonic day 15 metanephroi into the omentum of adult hosts is followed by differentiation, growth, vascularization and function of the implants. Here we show that survival of rats with all native renal mass removed can be increased by prior metanephros transplantation and ureteroureterostomy. Excretion of urine formed by metanephroi is prerequisite for enhanced survival. This is the first demonstration that life can be extended following de novo renal organogenesis.     

Genetic diversity of Dekkera bruxellensis yeasts isolated from Australian wineries

Yeasts of the genus Dekkera and its anamorph Brettanomyces represent a significant spoilage issue for the global wine industry. Despite this, there is limited knowledge of genetic diversity and strain distribution within wine and winery-related environments. In this study, amplified fragment length polymorphism (AFLP) analysis was conducted on 244 Dekkera bruxellensis isolates from red wine made in 31 winemaking regions of Australia. The results indicated there were eight genotypes among the isolates, and three of these were commonly found across multiple winemaking regions. Analysis of 26S rRNA gene sequences provided further evidence of three common, conserved groups, whereas a phylogeny based upon the AFLP data demonstrated that the most common D. bruxellensis genotype (I) in Australian red wine was highly divergent from the D. bruxellensis type strain (CBS 74).     

Using the Threat Probability Task to Assess Anxiety and Fear During Uncertain and Certain Threat

Fear of certain threat and anxiety about uncertain threat are distinct emotions with unique behavioral, cognitive-attentional, and neuroanatomical components. Both anxiety and fear can be studied in the laboratory by measuring the potentiation of the startle reflex. The startle reflex is a defensive reflex that is potentiated when an organism is threatened and the need for defense is high. The startle reflex is assessed via electromyography (EMG) in the orbicularis oculi muscle elicited by brief, intense, bursts of acoustic white noise (i.e., “startle probes”). Startle potentiation is calculated as the increase in startle response magnitude during presentation of sets of visual threat cues that signal delivery of mild electric shock relative to sets of matched cues that signal the absence of shock (no-threat cues). In the Threat Probability Task, fear is measured via startle potentiation to high probability (100% cue-contingent shock; certain) threat cues whereas anxiety is measured via startle potentiation to low probability (20% cue-contingent shock; uncertain) threat cues. Measurement of startle potentiation during the Threat Probability Task provides an objective and easily implemented alternative to assessment of negative affect via self-report or other methods (e.g., neuroimaging) that may be inappropriate or impractical for some researchers. Startle potentiation has been studied rigorously in both animals (e.g., rodents, non-human primates) and humans which facilitates animal-to-human translational research. Startle potentiation during certain and uncertain threat provides an objective measure of negative affective and distinct emotional states (fear, anxiety) to use in research on psychopathology, substance use/abuse and broadly in affective science. As such, it has been used extensively by clinical scientists interested in psychopathology etiology and by affective scientists interested in individual differences in emotion.     

“Unblocking” Serum Activity <Emphasis Type="Italic">in vitro</Emphasis> in the Polyoma System may Correlate with Antitumour Effects of Antiserum <Emphasis Type="Italic">in vivo</Emphasis>

In a variety of tumour systems, individuals carrying progressively growing neoplasms have lymphoid cells with a specific cytotoxic effect on cultured tumour cells from the same individual^1–4. Since the sera of tumour-bearing individuals have been shown to prevent tumour cell destruction by immune lymphocytes in vitro^2,5–8 and since this serum blocking activity appears early in primary and transplant tumour development^5,7, it has been suggested that the appearance of this serum blocking activity might be responsible for the progressive growth of tumours in individuals having cytotoxic lymphocytes. Counteraction of this blocking activity would thus be of primary importance in facilitating the function of an already existing or bolstered cell-mediated immunity. The serum blocking activity might be inhibited in various ways, by preventing the formation of blocking antibody or by interfering with its action (“unblocking”), as demonstrated in Moloney sarcoma regressor sera⁹. This type of serum also has a therapeutic effect on Moloney sarcomas in vivo^10,11, which has been tentatively attributed to its unblocking activity^8,9 or, possibly, to a complement-dependent cytotoxicity¹⁰. Tumour growth in the Moloney sarcoma system, however, might be due in part to continuous recruitment of neoplastic cells by virus-induced transformation and so the therapeutic effect could be due to a virus-neutralizing serum activity^9,10.