Therapeutic insulins and their large-scale manufacture

Biotechnological innovations over the past 25 years have underpinned the rapid development of a thriving biopharmaceutical sector. Therapeutic insulin remains one of the most commonly used products of pharmaceutical biotechnology and insulin-based products command annual global sales in excess of $4.5 billion. Innovations in its method of production and in particular the advent of engineered insulin analogues provide a fascinating insight into how scientific and technological advances have impacted upon the pharmaceutical biotechnology sector as a whole. Current insulin-based diabetes research is increasingly focused not on the insulin molecule per se, but upon areas such as the development of non-parenteral insulin delivery systems, as well as organ-/cell-based and gene therapy-based approaches to controlling the disease.     

Elucidating the substrate specificity and condensation domain activity of FkbP, the FK520 pipecolate-incorporating enzyme

Rapamycin, FK506, and FK520 are potent immunosuppressant natural product macrocycles generated by hybrid polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS) systems in streptomycetes. An important functional element within these molecules is an l-pipecolate moiety that is incorporated into the completed polyketide chain by the action of RapP/FkbP, a four-domain NRPS that also putatively serves to cyclize the chain after amino acid insertion. Here we report the expression and purification of recombinant FkbP from the FK520 biosynthetic pathway. Using a combination of radioassays and Fourier transform mass spectrometry, we demonstrate that once FkbP has been phosphopantetheinylated in vitro, its peptidyl carrier protein domain can be successfully loaded with l-pipecolic acid and, to a lesser extent, l-proline. The first condensation domain of FkbP is shown to be active through the successful acetylation of aminoacyl-S-FkbP using the appropriately loaded terminal acyl carrier protein from the PKS array, FkbA, as the chain donor. Site-directed mutagenesis confirmed that the N-terminal condensation domain catalyzes the transfer reaction. Acetylation of prolyl-S-FkbP was more rapid and occurred to a greater extent than that of pipecolyl-S-FkbP, a trend which was also observed with alternative acyl chain donors. These observations suggest that the adenylation domain of FkbP serves as the primary selectivity filter for pipecolate incorporation.     

PSORTb v.2.0: expanded prediction of bacterial protein subcellular localization and insights gained from comparative proteome analysis

MOTIVATION: PSORTb v.1.1 is the most precise bacterial localization prediction tool available. However, the program's predictive coverage and recall are low and the method is only applicable to Gram-negative bacteria. The goals of the present work are as follows: increase PSORTb's coverage while maintaining the existing precision level, expand it to include Gram-positive bacteria and then carry out a comparative analysis of localization. RESULTS: An expanded database of proteins of known localization and new modules using frequent subsequence-based support vector machines was introduced into PSORTb v.2.0. The program attains a precision of 96% for Gram-positive and Gram-negative bacteria and predictive coverage comparable to other tools for whole proteome analysis. We show that the proportion of proteins at each localization is remarkably consistent across species, even in species with varying proteome size. AVAILABILITY: Web-based version: http://www.psort.org/psortb. Standalone version: Available through the website under GNU General Public License. CONTACT: psort-mail@sfu.ca, brinkman@sfu.ca SUPPLEMENTARY INFORMATION: http://www.psort.org/psortb/supplementaryinfo.html.     

Synthesis and inhibitory activity of benzoic acid and pyridine derivatives on influenza neuraminidase

Based upon the activity and X-ray crystallographic studies of tri-substituted benzene derivatives containing carboxylic acid, acetamido and guanidine groups, we investigated the effect of the fourth substituent to fulfill the fourth pocket of neuraminidase enzyme. The groups selected as fourth substituents were hydroxymethyl, hydroxyethyl, oxime and amino. These tetra-substituted benzene derivatives were synthesized and evaluated for neuraminidase inhibitory activity. All these compounds were found to have poorer IC(50) values than the tri-substituted compounds. Further, benzene ring was replaced by pyridine ring and di, tri and tetra-substituted pyridine derivatives were synthesized. The activity of the pyridine derivatives was comparable to benzene derivatives. The fourth substituent seems to disturb the binding of the other three substituents, so the activity is reduced as compared to tri-substituted benzene and pyridine derivatives.     

The role of biphasic shocks for transthoracic cardioversion of atrial fibrillation

The modern generation of transthoracic defibrillators now employ impedance compensated biphasic waveforms. These new devices are superior to those with monophasic waveforms and practice is currently switching to biphasic defibrillators for the treatment of both ventricular and atrial fibrillation. However, there is no universal guideline for the use of biphasic defibrillators in direct current cardioversion of atrial fibrillation. This article reviews the use of biphasic defibrillation waveforms for transthoracic cardioversion of atrial fibrillation.     

Mechanism of eosinophil induced signaling in cholinergic IMR-32 cells

Eosinophils interact with nerve cells, leading to changes in neurotransmitter release, altered nerve growth, and protection from cytokine-induced apoptosis. In part, these interactions occur as a result of activation of neural nuclear factor (NF)-kappaB, which is activated by adhesion of eosinophils to neural intercellular adhesion molecule-1 (ICAM-1). The mechanism and consequence of signaling after eosinophil adhesion to nerve cells were investigated. Eosinophil membranes, which contain eosinophil adhesion molecules but not other eosinophil products, were coincubated with IMR-32 cholinergic nerve cells. The studies showed that there were two mechanisms of activation of NF-kappaB, one of which was dependent on reactive oxygen species, since it was inhibited with diphenyleneiodonium. This occurred at least 30 min after coculture of eosinophils and nerves. An earlier phase of NF-kappaB activation occurred within 2 min of eosinophil adhesion and was mediated by tyrosine kinase-dependent phosphorylation of interleukin-1 receptor-associated kinase-1 (IRAK-1). Coimmunoprecipitation experiments showed that both extracellular signal-regulated kinase 1/2 and IRAK-1 were recruited to ICAM-1 rapidly after coculture with eosinophil membranes. This was accompanied by an induction of ICAM-1, which was mediated by an IRAK-1-dependent pathway. These data indicate that adhesion of eosinophils to IMR-32 nerves via ICAM-1 leads to important signaling events, mediated via IRAK-1, and these in turn lead to expression of adhesion molecules.     

Infant learning: music and the baby brain

When it comes to listening to music, infants literally have a more open mind than their parents. Studies which investigate listening behaviour of babies and adults have shown that, as we learn to discriminate the musical sounds in our own environment, we become less sensitive to those of other cultures.     

Alien fly populations established at two Antarctic research stations

The populations of two non-native Dipterans have been established at two Antarctic research stations since at least 1998. Both belong to Sciaridae (black fungus midge), and have been determined to the genus Lycoriella. At Rothera Research Station, Antarctic Peninsula, flies are present in the station alcohol bond store, while at Casey Station, on the coast of continental Antarctica, a second Lycoriella sp. is found breeding in the station sewage facilities. Neither species is thought capable of surviving outside the protected environment of the research station buildings, but their establishment highlights the need for strict quarantine controls in order for National Operators in the Antarctic to conform to the Environmental Protocol of the Antarctic Treaty and prevent the introduction of alien species into Antarctica. Protocols for fly eradication are currently being implemented.