Directional cloning of native PCR products with preformed sticky ends (Autosticky PCR)

A novel method for the directional cloning of native PCR products was developed. Abasic sites in DNA templates make DNA polymerases stall at the site during synthesis of the complementary strand. Since the 5′ ends of PCR product strands contain built-in amplification primers, abasic sites within the primers result in the formation of 5′ single-stranded overhangs at the ends of the PCR product, enabling its direct ligation to a suitably cleaved cloning vector without any further modification. This “autosticky PCR” (AS-PCR) overcomes the problems caused by end sensitivity of restriction enzymes, or internal restriction sites within the amplified sequences, and enables the generation of essentially any desired 5′ overhang. Received: 11 August 1998 / Accepted: 2 October 1998     

The hprK gene of Enterococcus faecalis encodes a novel bifunctional enzyme: the HPr kinase/phosphatase

The HPr kinase of Gram-positive bacteria is an ATP-dependent serine protein kinase, which phosphorylates the HPr protein of the bacterial phosphotransferase system (PTS) and is involved in the regulation of carbohydrate metabolism. The hprK gene from Enterococcus faecalis was cloned via polymerase chain reaction (PCR) and sequenced. The deduced amino acid sequence was confirmed by microscale Edman degradation and mass spectrometry combined with collision-induced dissociation of tryptic peptides derived from the HPr kinase of E. faecalis . The gene was overexpressed in Escherichia coli , which does not contain any ATP-dependent HPr kinase or phosphatase activity. The homogeneous recombinant protein exhibits the expected HPr kinase activity as well as a P-Ser-HPr phosphatase activity, which was assumed to be a separate enzyme activity. The bifunctional HPr kinase/phosphatase acts preferentially as a kinase at high ATP levels of 2 mM occurring in glucose-metabolizing Streptococci . At low ATP levels, the enzyme hydrolyses P-Ser-HPr. In addition, high concentrations of phosphate present under starvation conditions inhibit the HPr kinase activity. Thus, a putative function of the enzyme may be to adjust the ratio of HPr and P-Ser-HPr according to the metabolic state of the cell; P-Ser-HPr is involved in carbon catabolite repression and regulates sugar uptake via the phosphotransferase system (PTS). Reinvestigation of the previously described Bacillus subtilis HPr kinase revealed that it also possesses P-Ser-HPr phosphatase activity. However, contrary to the E. faecalis enzyme, ATP alone was not sufficient to switch the phosphatase activity of the B. subtilis enzyme to the kinase activity. A change in activity of the B. subtilis HPr kinase was only observed when fructose-1,6-bisphosphate was also present.     

A multiscale mathematical model of cancer, and its use in analyzing irradiation therapies

Background  

Radiotherapy outcomes are usually predicted using the Linear Quadratic model. However, this model does not integrate complex features of tumor growth, in particular cell cycle regulation.     

Asymmetric inhibition by phlorizin of halide movements across the red blood cell membrane

Using human red cell ghosts, it was shown that external phlorizin inhibits Cl⁻ and I⁻ equilibrium exchange. Internal phlorizin has little effect on I⁻ exchange and no detectable effect on Cl⁻ exchange. This asymmetry is similar to that observed with the much more slowly exchanging SO₄²⁻ and different from that of sugars like l-arabinose or d-xylose which are inhibited by phlorizin from either surface.     

How intelligent is a cephalopod? Lessons from comparative cognition

The soft-bodied cephalopods including octopus, cuttlefish, and squid are broadly considered to be the most cognitively advanced group of invertebrates. Previous research has demonstrated that these large-brained molluscs possess a suite of cognitive attributes that are comparable to those found in some vertebrates, including highly developed perception, learning, and memory abilities. Cephalopods are also renowned for performing sophisticated feats of flexible behaviour, which have led to claims of complex cognition such as causal reasoning, future planning, and mental attribution. Hypotheses to explain why complex cognition might have emerged in cephalopods suggest that a combination of predation, foraging, and competitive pressures are likely to have driven cognitive complexity in this group of animals. Currently, it is difficult to gauge the extent to which cephalopod behaviours are underpinned by complex cognition because many of the recent claims are largely based on anecdotal evidence. In this review, we provide a general overview of cephalopod cognition with a particular focus on the cognitive attributes that are thought to be prerequisites for more complex cognitive abilities. We then discuss different types of behavioural flexibility exhibited by cephalopods and, using examples from other taxa, highlight that behavioural flexibility could be explained by putatively simpler mechanisms. Consequently, behavioural flexibility should not be used as evidence of complex cognition. Fortunately, the field of comparative cognition centres on designing methods to pinpoint the underlying mechanisms that drive behaviours. To illustrate the utility of the methods developed in comparative cognition research, we provide a series of experimental designs aimed at distinguishing between complex cognition and simpler alternative explanations. Finally, we discuss the advantages of using cephalopods to develop a more comprehensive reconstruction of cognitive evolution.