Serpins : form, function and dysfunction

The serpin superfamily of serine protease inhibitors is one of the most ubiquitous and successful classes of inhibitors in the living world. Their unique mechanism of suicide inhibition has led to much research and several important discoveries. They function via rapid incorporation of a reactive centre loop (RCL) within a β-sheet following the former's proteolysis by the target protease: the serpin thus achieves a conformation which is more stable than the native form. Through this conformational change, the target protease structure is distorted and its function disrupted. Alpha-1-antitrypsin (AAT) has often been studied as an archetype for the serpin superfamily, and is discussed in more detail in this review. Of particular interest are the mutant variants of AAT, which have a tendency to polymerise, and thus offer insights into some mechanisms of serpin polymerisation.peer-reviewe

The structure of protein molecules : in celebration of the International Year of Crystallography, 2014

Many people, including laymen, are aware of the double helical nature of the DNA molecule. A few may actually realise that it was the technique of X-ray crystallography that was the key to solving this structure. Even fewer will understand the uses and applications of crystallography to the most diverse of biological materials; proteins. In this review we discuss the application of a number of methodologies required to progress from a cloned gene to protein expression and purification, crystallisation conditions and eventually to X-ray structure determination. We provide our own experience in the field as examples of the procedures required. Protein crystallographers worldwide are contributing to our understanding of how enzymes work, how our immune system defends us against viruses and are using structural information to design novel pharmaceutical reagents.peer-reviewe

GST fusion protein expression vector for in-frame cloning and site directed mutagenesis

A novel glutathione S-transferase (GST) fusion protein expression vector, pGHX(-), was constructed to include a bacteriophage f1 origin of replication and a unique SalI restriction site. The former enables the packaging of the vector into filamentous bacteriophage for the isolation of single-stranded (ss)DNA, while the latter was engineered to form part of the DNA that encodes the protease recognition site for factor Xa, downstream of the gene encoding GST (Figure 1). These modifications allow both site-directed mutagenesis and the subsequent expression of the encoded GST fusion protein using a single plasmid. This obviates the need for subcloning mutated foreign genes from a single-stranded vector into an expression plasmid and therefore avoids many time-consuming protocols such as DNA digestion, fragment isolation, ligation, transformation and screening. Importantly, when the SalI site is used for blunt-end cloning, the amino acid sequence of the subsequently purified protein corresponds precisely and only to the encoding DNA of the cloned insert. No spurious N-terminal amino acids due to unavoidable cloning methodologies will be present in the purified protein product. This is particularly relevant to studies involving protein-protein interactions of purified binding domains and to studies of organelle protein targeting that involve N-terminal signal sequences.peer-reviewe

Role of protein structure in drug discovery

Many pharmaceuticals currently available were discovered either during the screening of natural of synthetic product libraries or by serendipitous observation. Such a \random" approach entails testing numerous compounds and developing countless high-throughput screening assays. On the other hand, a "rational" approach involves the structure-based route to drug discovery, where the structure of a target protein is determined. Hypothetical ligands may be predicted by molecular modelling, while movement of a molecule may be predicted by Molecular Dynamics Simulations prior to synthetic chemical synthesis of a particular molecule. Here, we will be discussing protein structure-based approaches to drug discovery.peer-reviewe

Master Franchising as an Entry Strategy: Marketing and Legal Implications

In this paper the authors investigate the establishment of franchise agreements as a viable alternative to enter a foreign market. Specifically, the spotlight is on the strategy called master franchising. The authors first review the concept of franchising and identify master franchising as a strategic option. Next, they focus on the mechanics of structuring the required agreements. Last, the authors explore strategies, trends, and current opportunities and limitations of international franchising

Cloning, expression and characterisation of two manganese superoxide dismutases from Caenorhabditis elegans

Two genes encoding manganese superoxide dismutase (sod-2 and sod-3) have been identified in the nematode Caenorhabditis elegans. Each gene is composed of five exons, and intron positions are identical; however, intron sizes and sequences are not the same. The predicted protein sequences are 86.3% homologous (91.8% conservative), and the cDNAs are only 75.2% homologous. Both deduced protein sequences contain the expected N-terminal mitochondrial transit peptides. Reverse transcriptase polymerase chain reaction analysis shows that both genes are expressed under normal growth conditions and that their RNA transcripts aretrans-spliced to the SL-1 leader sequence. The latter result together with Northern blot analysis indicate that both genes have mono-cistronic transcripts. The sod-3 gene was mapped to chromosome X, and the location of sod-2 was confirmed to be chromosome I. Polymerase chain reaction was used to amplify the cDNA regions encoding the predicted mature manganese superoxide dismutase proteins and each was cloned and expressed to high levels in Escherichia coli cells deficient in cytosolic superoxide dismutases. Both proteins were shown to be active in E. coli, providing similar protection against methyl viologen-induced oxidative stress. The expressed enzymes, which were not inhibited by hydrogen peroxide or cyanide, are dimeric, show quite different electrophoretic mobilities and isoelectric points, but exhibit comparable specific activities.peer-reviewe

In vivo transcription of ribosomal RNA in relation to the mitotic cycle in Physarum polycephalum

We have investigated the transcription of ribosomal RNA in plasmodia of Physarum polycephalum by a combination of pulse-labelling with [3H]uridine and RNA; DNA hybridization. The DNA used for the hybridization was a Hindlll restriction fragment (cloned in bacteriophage λ) of Physarum ribosomal DNA that carries a substantial fraction of the rRNA genes, enabling the ribosomal transcripts in the newly synthesized RNA to be measured. We found that ribosomal RNA constituted about 40 % of the pulse-labelled RNA at all times during the synchronous mitotic cycle.peer-reviewe

Carbon Dioxide Gas Sensors and Method of Manufacturing and Using Same

A gas sensor includes a substrate and a pair of interdigitated metal electrodes selected from the group consisting of Pt, Pd, Au, Ir, Ag, Ru, Rh, In, and Os. The electrodes each include an upper surface. A first solid electrolyte resides between the interdigitated electrodes and partially engages the upper surfaces of the electrodes. The first solid electrolyte is selected from the group consisting of NASICON, LISICON, KSICON, and .beta.''-Alumina (beta prime-prime alumina in which when prepared as an electrolyte is complexed with a mobile ion selected from the group consisting of Na.sup.+, K.sup.+, Li.sup.+, Ag.sup.+, H.sup.+, Pb.sup.2+, Sr.sup.2+ or Ba.sup.2+). A second electrolyte partially engages the upper surfaces of the electrodes and engages the first solid electrolyte in at least one point. The second electrolyte is selected from the group of compounds consisting of Na.sup.+, K.sup.+, Li.sup.+, Ag.sup.+, H.sup.+, Pb.sup.2+, Sr.sup.2+ or Ba.sup.2+ ions or combinations thereof

Purification, crystallization and X-ray structures of the two manganese superoxide dismutases from Caenorhabditis elegans

Two manganese superoxide dismutase enzymes isolated from the eukaryote C. elegans have been characterized and their structures determined. The closely related structures reveal a striking similarity to manganese superoxide dismutase found in humans