In vitro construction of gshB::kan in Escherichia coli and use of gshB::kan in mapping the gshB locus.

The Escherichia coli structural gene for glutathione synthetase, gshB, was cloned into pBR322. Plasmids containing gshB were able to complement the glutathione requirement of a trxA gshB double mutant, and cells containing the plasmids were found to have elevated levels of glutathione synthetase. A mutant gshB allele was constructed by inserting the kan gene from pUC4K into a unique HpaI site located within gshB. The resulting plasmid-encoded allele was used to replace a genomic gshB+ by homologous recombination. The resulting strain had no detectable glutathione synthetase activity. The gshB allele containing the kan insertion was used to map gshB on the E. coli chromosome by P1 transduction. The results indicated that gshB is located at 63.4 min, between metK and speC. The allele was further localized to a region of 3,100 to 3,120 kilobase pairs on the physical map (restriction map) of E. coli by DNA-DNA hybridization to a series of lambda bacteriophages (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987).     

Antithrombin: in control of coagulation

Antithrombin is a serine proteinase inhibitor (serpin) which controls the process of coagulation. It has a well defined structure, consisting of three beta-sheets, nine alpha-helices and a reactive centre loop (RCL). The RCL contains the reactive centre which harbours a bait sequence for target proteases; cleavage results in inhibition by a unique mechanism. The inhibitory activity of antithrombin is controlled by its interaction with the co-factor, heparin, which accelerates its interaction with target proteases. This ensures that heparin and its newer derivatives, such as heparin pentasaccharide, are the mainstay therapeutics for control of thrombosis or inappropriate clotting. The clinical importance of antithrombin is manifested by its clear association with thrombosis when deficiency states occur.     

Focus: Rare Disease: Neuromonitoring in Rare Disorders of Metabolism

Inborn errors of metabolism (IEM) are a unique class of genetic diseases due to mutations in genes involved in key metabolic pathways. The combined incidence of IEM has been estimated to be as high as 1:1000. Urea Cycle disorders (UCD), one class of IEM, can present with cerebral edema and represent a possible target to explore the utility of different neuromonitoring techniques during an hyperammonemic crisis. The last two decades have brought advances in the early identification and comprehensive management of UCD, including further understanding of neuroimaging patterns associated with neurocognitive function. Nonetheless, very important questions remain about the potential acute neurotoxic effects of hyperammonemia to better understand how to treat and prevent secondary brain injury. In this review, we describe existing neuromonitoring techniques that have been used in rare metabolic disorders to assess and allow amelioration of ongoing brain injury. Directions of future research should be focused on identifying new diagnostic approaches in the management of metabolic crises to optimize care and reduce long term morbidity and mortality in patients with IEM.     

Expression of a CaMV::sak-gusA-mgfp gene in tobacco plants

A gene encoding staphylokinase from Staphylococcus aureus was cloned into the plant transformation binary vector pCAMBIA1303. The presence of a CaMV::sak-gusA-mgfp gene in Agrobacterium was confirmed by polymerase chain reaction PCR. Tobacco seedlings were used as explants for Agrobacterium tumefaciens-mediated transformation with the pCAMBIA1303sak vector carrying the fusion gene construct CaMV::sak-gusA-mgfp and the expression of the fusion gene was identified in Nicotiana tabacum plants by β-glucuronidas assay. This revised version was published online in August 2006 with corrections to the Cover Date.     

Mini-review: Osteoblasts: An in vitro model of bone-implant interactions

Well-characterized osteoblasts provide a successful in vitro model to study bone-biomaterial interactions. Knowledge of the events occurring at this tissue-biomaterial interface could lead to the design of improved orthopedic/dental biomaterials which elicit specific and desirable responses from surrounding cells/tissues, optimize function of osteoblasts (the boneforming cells), and enhance long-term bone-implant bonding. (c) 1994 John Wiley & Sons, Inc.     

Perspectives: Hamilton's Legacy: Mechanisms of Kin Recognition in Humans

The behavior literature is replete with examples of individuals exhibiting costly acts that benefit someone else. These examples troubled Darwin so much so that he thought they would be fatal to his theory of natural selection. A century later, W. D. Hamilton refined that theory by showing, quantitatively, that such acts could be favored if the individuals involved were relatives. His theory of inclusive fitness is generally considered one of the greatest theoretical advances in evolution since Darwin's time. Less appreciated from Hamilton's 1964 paper is the hypothesis that mechanisms favoring accurate kin recognition will also be selected. Here, I review those recognition mechanisms and survey the literature on human kin recognition. Although not often considered, humans both produce cues to kinship that vary with genetic relatedness and have perceptual abilities to detect these cues in others and assess that relatedness. The potential functions of these abilities are discussed. Importantly, gaps in our understanding of the development and use of recognition mechanisms are noted.     

Functional consequences of single:double ring transitions in chaperonins: life in the cold

The cpn60 and cpn10 genes from psychrophilic bacterium, Oleispira antarctica RB8, showed a positive effect in Escherichia coli growth at low temperature, shifting its theoretical minimal growth temperature from +7.5 degrees C to -13.7 degrees C [Ferrer, M., Chernikova, T.N., Yakimov, M., Golyshin, P.N., and Timmis, K.N. (2003) Nature Biotechnol 21: 1266-1267]. To provide experimental support for this finding, Cpn60 and 10 were overproduced in E. coli and purified to apparent homogeneity. Recombinant O.Cpn60 was identical to the native protein based on tetradecameric structure, and it dissociates during native PAGE. Gel filtration and native PAGE revealed that, in vivo and in vitro, (O.Cpn60)(7) was the active oligomer at 4-10 degrees C, whereas at > 10 degrees C, this complex was converted to (O.Cpn60)(14). The dissociation reduces the ATP consumption (energy-saving mechanism) and increases the refolding capacity at low temperatures. In order for this transition to occur, we demonstrated that K468 and S471 may play a key role in conforming the more advantageous oligomeric state in O.Cpn60. We have proved this hypothesis by showing that single and double mutations in K468 and S471 for T and G, as in E.GroEL, produced a more stable double-ring oligomer. The optimum temperature for ATPase and chaperone activity for the wild-type chaperonin was 24-28 degrees C and 4-18 degrees C, whereas that for the mutants was 45-55 degrees C and 14-36 degrees C respectively. The temperature inducing unfolding (T(M)) increased from 45 degrees C to more than 65 degrees C. In contrast, a single ring mutant, O.Cpn60(SR), with three amino acid substitutions (E461A, S463A and V464A) was as stable as the wild type but possessed refolding activity below 10 degrees C. Above 10 degrees C, this complex lost refolding capacity to the detriment of the double ring, which was not an efficient chaperone at 4 degrees C as the single ring variant. We demonstrated that expression of O.Cpn60(WT) and O.Cpn60(SR) leads to a higher growth of E. coli at 4 degrees C ( micro (max), 0.22 and 0.36 h(-1) respectively), whereas at 10-15 degrees C, only E. coli cells expressing O.Cpn60 or O.Cpn60(DR) grew better than parental cells (-cpn). These results clearly indicate that the single-to-double ring transition in Oleispira chaperonin is a wild-type mechanism for its thermal acclimation. Although previous studies have also reported single-to-double ring transitions under many circumstances, this is the first clear indication that single-ring chaperonins are necessary to support growth when the temperature falls from 37 degrees C to 4 degrees C.