Stringent and reproducible tetracycline-regulated transgene expression by site-specific insertion at chromosomal loci with pre-characterised induction characteristics

Background  

The ability to regulate transgene expression has many applications, mostly concerning the analysis of gene function. Desirable induction characteristics, such as low un-induced expression, high induced expression and limited cellular heterogeneity, can be seriously impaired by chromosomal position effects at the site of transgene integration. Many clones may therefore need to be screened before one with optimal induction characteristics is identified. Furthermore, such screens must be repeated for each new transgene investigated, and comparisons between clones with different transgenes is complicated by their different integration sites.     

An IGF-I promoter polymorphism modifies the relationships between birth weight and risk factors for cardiovascular disease and diabetes at age 36

BACKGROUND: Biochemical testing for pheochromocytoma by measurement of fractionated plasma metanephrines is limited by false positive rates of up to 18% in people without known genetic predisposition to the disease. The plasma normetanephrine fraction is responsible for most false positives and plasma normetanephrine increases with age. The objective of this study was to determine if we could improve the specificity of fractionated plasma measurements, by statistically adjusting for age. METHODS: An age-adjusted metanephrine score was derived using logistic regression from 343 subjects (including 33 people with pheochromocytoma) who underwent fractionated plasma metanephrine measurements as part of investigations for suspected pheochromocytoma at Mayo Clinic Rochester (derivation set). The performance of the age-adjusted score was validated in a dataset of 158 subjects (including patients 23 with pheochromocytoma) that underwent measurements of fractionated plasma metanephrines at Mayo Clinic the following year (validation dataset). None of the participants in the validation dataset had known genetic predisposition to pheochromocytoma. RESULTS: The sensitivity of the age-adjusted metanephrine score was the same as that of traditional interpretation of fractionated plasma metanephrine measurements, yielding a sensitivity of 100% (23/23, 95% confidence interval [CI] 85.7%, 100%). However, the false positive rate with traditional interpretation of fractionated plasma metanephrine measurements was 16.3% (22/135, 95% CI, 11.0%, 23.4%) and that of the age-adjusted score was significantly lower at 3.0% (4/135, 95% CI, 1.2%, 7.4%) (p < 0.001 using McNemar's test). CONCLUSION: An adjustment for age in the interpretation of results of fractionated plasma metanephrines may significantly decrease false positives when using this test to exclude sporadic pheochromocytoma. Such improvements in false positive rate may result in savings of expenditures related to confirmatory imaging.     

Distribution, genetic diversity, and variable expression of the gene encoding hyaluronate lyase within the Streptococcus suis population

Although Streptococcus suis is an economically important pathogen of pigs and an occasional cause of zoonotic infections of humans knowledge of crucial virulence factors, and as a consequence targets for therapeutic or prophylactic intervention, remains limited. Here we describe a detailed study of the distribution, diversity, and in vitro expression of hyaluronate lyase, a protein implicated as a virulence factor of many mucosal pathogens. The gene encoding hyaluronate lyase, hyl, was present in all 309 bona fide S. suis isolates examined representing diverse serotypes, geographic sources, and clinical backgrounds. Examination of the genetic diversity of hyl by RFLP and sequence analysis indicated a pattern of diversity shared by many gram-positive surface proteins with a variable 5' region encoding the most distal cell surface-exposed regions of the protein and a much more conserved 3' region encoding domains more closely associated with the bacterial cell. Variation occurs by several mechanisms, including the accumulation of point mutations and deletion and insertion events, and there is clear evidence that genetic recombination has contributed to molecular variation in this gene. Despite the ubiquitous presence of hyl, the corresponding enzyme activity was detected in fewer than 30% of the 309 isolates. In several cases this lack of activity correlates with the presence of mutations (either sequence duplications or point mutations) within hyl that result in a truncated polypeptide. There is a striking absence of hyaluronate lyase activity in a large majority of isolates from classic S. suis invasive disease, indicating that this protein is probably not a crucial virulence factor, although activity is present in significantly higher numbers of isolates associated with pneumonia.     

Fluorescent reagents for in vitro studies of lipid-linked steps of bacterial peptidoglycan biosynthesis: derivatives of UDPMurNAc-pentapeptide containing d-cysteine at position 4 or 5

UDPMurNAc-L-Ala-gamma-D-Glu-X-D-Ala-DAla (X = L-Lys or m-DAP) is the cytoplasmic precursor for the lipid-linked cycle of bacterial peptidoglycan biosynthesis, consisting of at least four enzymatic reactions, which are targets for antibacterial agents. Fluorescent derivatives of the UDPMurNAc-pentapeptide labelled at the 3rd, 4th, and 5th position of the peptide chain were prepared chemoenzymatically, in order to study the reactions catalysed by enzymes in this cycle. Derivatives labelled on the epsilon-amino group of the 3rd amino acid (N-dansyl, N-fluorescamine and N-phthalaldehyde) were prepared by chemical modification. Two methods were developed for preparation of analogues of UDPMurNAc-pentapeptide containing D-cysteine at position 4 or 5: either by MurF-catalysed ligation of the UDPMurNAc-tripeptide to synthetic D-Ala-D-Cys or D-Cys-D-Ala dipeptides; or by enzymatic synthesis of D-Ala-D-Cys by ligase VanD. D-Cys-containing UDPMurNAc-pentapeptides were labelled with pyrene maleimide, to give 4-pyrene and 5-pyrene labelled derivatives. The fluorescent UDPMurNAc-pentapeptides were processed as substrates by Escherichia coli MraY or E. coli membranes, giving 1.5-150-fold changes in fluorescence upon transformation to lipid intermediate I. Subsequent processing to lipid intermediate II gave rise only to small changes in fluorescence. Pyrene-labelled lipid intermediates I and II can be generated using Micrococcus flavus membranes, enabling the study of the later lipid-linked steps.     

Tribological characteristics of healthy tendon

Tendons transfer muscular forces efficiently and painlessly, facilitating joint motion. Whilst the tribology of articular cartilage is constantly explored, a poorer understanding remains of tendon lubrication and friction. This study reports experimental data describing the tribological characteristics of tendon and its surrounding tissue, before presenting an arithmetic solution to facilitate numerical modelling. The experimental characteristics of the tensile (i.e. mid-substance) and compressive (i.e. fibrocartilaginous) regions of bovine flexor tendon were investigated using a pin-on-plate tribometer, with immunofluroscence analysis describing the relative intensity and distribution of surface-bound lubricin. Arithmetic analysis considering the digital extensor tendon determined that, in physiological conditions, the tensile tendon region was able to generate elastohydrodynamic lubrication (EHL). The equivalent region of compressive tendon exhibited a higher intensity of surface-bound lubricin which, it is hypothesised, serves to minimise the increased frictional resistance due to generating only mixed or boundary lubrication regimes. Arithmetic analysis indicates that, given a more favourable biomechanical environment, this region can also generate EHL. Whilst acknowledging the limitations of transferring data from an animal model to a clinical environment, by providing the first data and equations detailing the film thicknesses and lubrication regime for these two tendon regions it is hoped that clinicians, engineers and scientists can consider improved clinical strategies to tackle both tendinopathy and tendon rupture.     

Kinetic characterization of lipid II-Ala:alanyl-tRNA ligase (MurN) from Streptococcus pneumoniae using semisynthetic aminoacyl-lipid II substrates

MurM and MurN are tRNA-dependent ligases that catalyze the addition of the first (L-Ala/L-Ser) and second (L-Ala) amino acid onto lipid II substrates in the biosynthesis of the peptidoglycan layer of Streptococcus pneumoniae. We have previously characterized the first ligase, MurM (Lloyd, A. J., Gilbey, A. M., Blewett, A. M., De Pascale, G., El Zoeiby, A., Levesque, R. C., Catherwood, A. C., Tomasz, A., Bugg, T. D., Roper, D. I., and Dowson, C. G. (2008) J. Biol. Chem. 283, 6402-6417). In order to characterize the second ligase MurN, we have developed a chemoenzymatic route to prepare the lipid II-Ala and lipid II-Ser substrates. Recombinant MurN enzymes from penicillin-resistant (159) and -sensitive (Pn16) S. pneumoniae were expressed and purified as MBP fusion proteins and reconstituted using a radiochemical assay. MurN ligases from strains 159 and Pn16 both showed a 20-fold higher catalytic efficiency for lipid II-L-Ala over lipid II-l-Ser, with no activity against unmodified lipid II, and similar kinetic parameters were measured for MurN from penicillin-resistant and penicillin-sensitive strains. These results concur with the peptidoglycan analysis of S. pneumoniae, in which the major cross-link observed is L-Ala-L-Ala. The combined action of ligases MurM and MurN is therefore required in order to rationalize the high level of dipeptide cross-links in penicillin-resistant S. pneumoniae, with ligase MurM showing the major difference between penicillin-resistant and penicillin-sensitive strains.     

Characterization of tRNA-dependent peptide bond formation by MurM in the synthesis of Streptococcus pneumoniae peptidoglycan

MurM is an aminoacyl ligase that adds l-serine or l-alanine as the first amino acid of a dipeptide branch to the stem peptide lysine of the pneumococcal peptidoglycan. MurM activity is essential for clinical pneumococcal penicillin resistance. Analysis of peptidoglycan from the highly penicillin-resistant Streptococcus pneumoniae strain 159 revealed that in vivo and in vitro, in the presence of the appropriate acyl-tRNA, MurM(159) alanylated the peptidoglycan epsilon-amino group of the stem peptide lysine in preference to its serylation. However, in contrast, identical analyses of the penicillin-susceptible strain Pn16 revealed that MurM(Pn16) activity supported serylation more than alanylation both in vivo and in vitro. Interestingly, both MurM(Pn16) acylation activities were far lower than the alanylation activity of MurM(159). The resulting differing stem peptide structures of 159 and Pn16 were caused by the profoundly greater catalytic efficiency of MurM(159) compared with MurM(Pn16) bought about by sequence variation between these enzymes and, to a lesser extent, differences in the in vivo tRNA(Ala):tRNA(Ser) ratio in 159 and Pn16. Kinetic analysis revealed that MurM(159) acted during the lipid-linked stages of peptidoglycan synthesis, that the d-alanyl-d-alanine of the stem peptide and the lipid II N-acetylglucosaminyl group were not essential for substrate recognition, that epsilon-carboxylation of the lysine of the stem peptide was not tolerated, and that lipid II-alanine was a substrate, suggesting an evolutionary link to staphylococcal homologues of MurM such as FemA. Kinetic analysis also revealed that MurM recognized the acceptor stem and/or the TPsiC loop stem of the tRNA(Ala). It is anticipated that definition of the minimal structural features of MurM substrates will allow development of novel resistance inhibitors that will restore the efficacy of beta-lactams for treatment of pneumococcal infection.     

Multilocus Sequence Typing for Comparison of Veterinary and Human Isolates of Campylobacter jejuni

Multilocus sequence typing (MLST) has been applied to 266 Campylobacter jejuni isolates, mainly from veterinary sources, including cattle, sheep, poultry, pigs, pets, and the environment, as well as isolates from human cases of campylobacteriosis. The populations of veterinary and human isolates overlap, suggesting that most veterinary sources should be considered reservoirs of pathogenic campylobacters. There were some associations between source and sequence type complex, indicating that host or source adaptation may exist. The pig isolates formed a distinct group by MLST and may well represent a potential pig-adapted clone of C. jejuni. A subset (n = 82) of isolates was reanalyzed with a second MLST scheme which provided a unique set of isolates that had been analyzed at a total of 12 loci. The distribution of isolates among the complexes in each of the two schemes was similar but not identical. In addition to isolates from human outbreaks, one group of isolates that were not epidemiologically linked was also identical at all 12 loci. This group of isolates is believed to represent another stable strain of C. jejuni.     

Animal models for an enzymic blood-brain barrier mechanism for therapeutically administered L-dopa

Tissue slices from three brain regions of the rat and cat were incubated in a medium containing L-DOPA and nialamide, and studied by the formaldehyde-fluorescence method. Unlike the rat brain capillary walls, which showed a marked increase in fluorescence due to uptake of L-DOPA and subsequent dopamine formation and accumulation, the capillary walls of the cat brain samples did not show evidence of dopamine accumulation.The nature of a blood-brain barrier to L-DOPA in man is discussed with regard to this finding.