DNA decay and limited Rad53 activation after liquid holding of UV-treated nucleotide excision repair deficient S. cerevisiae cells

The DNA damage checkpoint is a surveillance mechanism activated by DNA lesions and devoted to the maintenance of genome stability. It is considered as a signal transduction cascade, involving a sensing step, the activation of a set of protein kinases and the transmission and amplification of the damage signal through several phosphorylation events. In budding yeast many players of this pathway have been identified. Recent work showed that G1 and G2 checkpoint activation in response to UV irradiation requires prior recognition and processing of UV lesions by nucleotide excision repair (NER) factors that likely recruit checkpoint proteins near the damage. However, another report suggested that NER was not required for checkpoint function. Since the functional relationship between repair mechanisms and checkpoint activation is a very important issue in the field, we analyzed, under different experimental conditions, whether lesion processing by NER is required for checkpoint activation. We found that DNA damage checkpoint can be triggered in an NER-independent manner only if cells are subjected to liquid holding after UV treatment. This incubation causes a time-dependent breakage of DNA strands in NER-deficient cells and leads to partial activation of the checkpoint kinase. The analysis of the genetic requirements for this alternative activation pathway suggest that it requires Mec1 and the Rad17 complex and that the observed DNA breaks are likely to be due to spontaneous decay of damaged DNA.     

Synthesis and biological activity of 2-aminothiazoles as novel inhibitors of PGE2 production in cells

This Letter presents the synthesis and biological evaluation of a collection of 2-aminothiazoles as a novel class of compounds with the capability to reduce the production of PGE(2) in HCA-7 human adenocarcinoma cells. A total of 36 analogs were synthesized and assayed for PGE(2) reduction, and those with potent cellular activity were counter screened for inhibitory activity against COX-2 in a cell free assay. In general, analogs bearing a 4-phenoxyphenyl substituent in the R(2) position were highly active in cells while maintaining negligible COX-2 inhibition. Specifically, compound 5l (R(1)=Me, R(2)=4-OPh-Ph, R(3)=CH(OH)Me) exhibited the most potent cellular PGE(2) reducing activity of the entire series (EC(50)=90 nM) with an IC(50) value for COX-2 inhibition of >5 μM in vitro. Furthermore, the anti-tumor activity of analog 1a was analyzed in xenograft mouse models exhibiting promising anti-cancer activity.     

The expression of a highly expressed Bacillus subtilis gene is not reduced by introduction of multiple codons normally not present in such genes

A recombinant DNA Proteus mirabilis L-form expression system, LVI (pJS127), was used to synthesize human fusion interferon alpha 1 (f-IFN-alpha 1). In the expression plasmid used, the complete coding sequence of IFN-alpha 1 was linked to the streptococcal speA promoter and the 5' end of the speA structural gene including its signal sequence coding region. LVI (pJS127) was capable of complete secretion into the culture medium of biologically active f-IFN-alpha 1 whose identity was confirmed by immunological and chemical evidence. In particular, bacterial L-forms were for the first time shown to be capable of correct signal peptide processing, as determined by N-terminal sequencing of the secreted f-IFN.     

Cathodes as electron donors for microbial metabolism: Which extracellular electron transfer mechanisms are involved?

This review illuminates extracellular electron transfer mechanisms that may be involved in microbial bioelectrochemical systems with biocathodes. Microbially-catalyzed cathodes are evolving for new bioprocessing applications for waste(water) treatment, carbon dioxide fixation, chemical product formation, or bioremediation. Extracellular electron transfer processes in biological anodes, were the electrode serves as electron acceptor, have been widely studied. However, for biological cathodes the question remains: what are the biochemical mechanisms for the extracellular electron transfer from a cathode (electron donor) to a microorganism? This question was approached by not only analysing the literature on biocathodes, but also by investigating known extracellular microbial oxidation reactions in environmental processes. Here, it is predicted that in direct electron transfer reactions, c-type cytochromes often together with hydrogenases play a critical role and that, in mediated electron transfer reactions, natural redox mediators, such as PQQ, will be involved in the bioelectrochemical reaction. These mechanisms are very similar to processes at the bioanode, but the components operate at different redox potentials. The biocatalyzed cathode reactions, thereby, are not necessarily energy conserving for the microorganism.     

G Protein-coupled receptor kinase 2 (GRK2): A novel modulator of insulin resistance

G protein-coupled receptor kinase 2 (GRK2) is emerging as a key, integrative node in many signalling pathways. Besides its canonical role in the modulation of the signalling mediated by many G protein-coupled receptors (GPCR), this protein can display a very complex network of functional interactions with a variety of signal transduction partners, in a stimulus, cell type, or context-specific way. We review herein recent data showing that GRK2 can regulate insulin-triggered transduction cascades at different levels and that this protein plays a relevant role in insulin resistance and obesity in vivo, what uncovers GRK2 as a potential therapeutic target in the treatment of these disorders.     

Molecular mechanics of mouse cardiac myosin isoforms

Two myosin isoforms are expressed in myocardium, alphaalpha-homodimers (V(1)) and betabeta-homodimers (V(3)). V(1) exhibits higher velocities and myofibrillar ATPase activities compared with V(3). We also observed this for cardiac myosin from normal (V(1)) and propylthiouracil-treated (V(3)) mice. Actin velocity in a motility assay (V(actin)) over V(1) myosin was twice that of V(3) as was the myofibrillar ATPase. Myosin's average force (F(avg)) was similar for V(1) and V(3). Comparing V(actin) and F(avg) across species for both V(1) and V(3), our laboratory showed previously (VanBuren P, Harris DE, Alpert NR, and Warshaw DM. Circ Res 77: 439-444, 1995) that mouse V(1) has greater V(actin) and F(avg) compared with rabbit V(1). Mouse V(3) V(actin) was twice that of rabbit V(actin). To understand myosin's molecular structure and function, we compared alpha- and beta-cardiac myosin sequences from rodents and rabbits. The rabbit alpha- and beta-cardiac myosin differed by eight and four amino acids, respectively, compared with rodents. These residues are localized to both the motor domain and the rod. These differences in sequence and mechanical performance may be an evolutionary attempt to match a myosin's mechanical behavior to the heart's power requirements.     

Auto‐phosphorylation of a voltage‐gated K+ channel controls non‐associative learning

Here, we characterize a new K⁺ channel–kinase complex that operates in the metazoan Caenorhabditis elegans to control learning behaviour. This channel is composed of a pore‐forming subunit, dubbed KHT‐1 (73% homology to human Kv3.1), and the accessory subunit MPS‐1, which shows kinase activity. Genetic, biochemical and electrophysiological evidence show that KHT‐1 and MPS‐1 form a complex in vitro and in native mechanosensory PLM neurons, and that KHT‐1 is a substrate for the kinase activity of MPS‐1. Behavioural analysis further shows that the kinase activity of MPS‐1 is specifically required for habituation to repetitive mechanical stimulation. Thus, worms bearing an inactive MPS‐1 variant (D178N) respond normally to touch on the body but do not habituate to repetitive mechanical stimulation such as tapping on the side of the Petri dish. Hence, the phosphorylation status of KHT‐1–MPS‐1 seems to be linked to distinct behavioural responses. In the non‐phosphorylated state the channel is necessary for the normal function of the touch neurons. In the auto‐phosphorylated state the channel acts to induce neuronal adaptation to mechanical stimulation. Taken together, these data establish a new mechanism of dynamic regulation of electrical signalling in the nervous system.