Purification and characterization of uracil phosphoribosyltransferase from Crithidia luciliae

1. Uracil phosphoribosyltransferase (UPRTase) was purified 370-fold from the protozoan parasite, Crithidia luciliae. 2. The enzyme was a dimer of mol. wt 80 000 and was highly specific for uracil. 3. GTP, which is an activator of UPRTase from E. coli had a slight inhibitory effect on the parasite enzyme. 4. The C. luciliae UPRTase demonstrated a broad specificity for activating divalent metal ions.     

2-(Trimethylammonium) Ethyl (R)-3-Methoxy-3-oxo-2-Stearamidopropyl Phosphate Suppresses Osteoclast Maturation and Bone Resorption by Targeting Macrophage-Colony Stimulating Factor Signaling

2-(Trimethylammonium) ethyl (R)-3-methoxy-3-oxo-2-stearamidopropyl phosphate [(R)-TEMOSPho], a derivative of an organic chemical identified from a natural product library, promotes highly efficient megakaryopoiesis. Here, we show that (R)-TEMOSPho blocks osteoclast maturation from progenitor cells of hematopoietic origin, as well as blocking the resorptive function of mature osteoclasts. The inhibitory effect of (R)-TEMOSPho on osteoclasts was due to a disruption of the actin cytoskeleton, resulting from impaired downstream signaling of c-Fms, a receptor for macrophage-colony stimulating factor linked to c-Cbl, phosphoinositol-3-kinase (PI3K), Vav3, and Rac1. In addition, (R)-TEMOSPho blocked inflammation-induced bone destruction by reducing the numbers of osteoclasts produced in mice. Thus, (R)-TEMOSPho may represent a promising new class of antiresorptive drugs for the treatment of bone loss associated with increased osteoclast maturation and activity.     

Mitochondrial DNA Copy Number in Peripheral Blood and Melanoma Risk

Mitochondrial DNA (mtDNA) copy number in peripheral blood has been suggested as risk modifier in various types of cancer. However, its influence on melanoma risk is unclear. We evaluated the association between mtDNA copy number in peripheral blood and melanoma risk in 500 melanoma cases and 500 healthy controls from an ongoing melanoma study. The mtDNA copy number was measured using real-time polymerase chain reaction. Overall, mean mtDNA copy number was significantly higher in cases than in controls (1.15 vs 0.99, P<0.001). Increased mtDNA copy number was associated with a 1.45-fold increased risk of melanoma (95% confidence interval: 1.12-1.97). Significant joint effects between mtDNA copy number and variables related to pigmentation and history of sunlight exposure were observed. This study supports an association between increased mtDNA copy number and melanoma risk that is independent on the known melanoma risk factors (pigmentation and history of sunlight exposure).     

The sequence specificity of alkylation for a series of benzoic acid mustard and imidazole-containing distamycin analogues: the importance of local sequence conformation.

The covalent sequence specificity of a series of nitrogen mustard and imidazole-containing analogues of distamycin was determined using modified sequencing techniques. The analogues tether benzoic acid mustard (BAM) and possess either one, two or three imidazole units. Examination of the alkylation specificity revealed that BAM produced guanine-N7 lesions in a pattern similar to conventional nitrogen mustards. The monoimidazole-BAM conjugate also produced guanine-N7 alkylation in a similar pattern to BAM, but at a 100-fold lower dose. The diimidazole and triimidazole conjugates did not produce detectable guanine-N7 alkylation but only alkylated at selected sites in the minor groove. Unexpectedly, the alkylation specificity at equivalent doses was nearly identical to that found for the previously reported pyrrole-BAM conjugates. The consensus sequence, 5'-TTTTGPuwas strongly alkylated by the triimidazole conjugate in preference to other similar sites including three occurrences of 5'-TTTTAA. Footprinting studies were carried out to examine the non-covalent DNA binding interactions. These studies revealed that the tripyrrole- BAM conjugate bound non-covalently to the same AT-rich sites as distamycin. In contrast, whereas the Im3lexitropsin bound non-covalently to GC-rich sequences, the triimidazole-BAM conjugate did not detectably footprint to either GC- or AT-rich regions at equivalent doses. The results indicate that the alkylation event is not solely dictated by the non-covalent binding and might be influenced by a unique sequence dependent conformational feature of the consensus sequence 5'-TTTTGPu.     

Metaphysics to the rescue?: Four-dimensionalism and the twinning argument against conceptionism

The view that human beings begin to exist at fertilization (namely conceptionism) faces a serious challenge from the twinning argument, that identical twins coming from the same zygote must be numerically distinct from the zygote and so did not exist at fertilization. Recently, some philosophers have claimed that the twinning argument rests on a particular metaphysical theory of persistence, namely endurantism, on which a human being, for example, is wholly present at every moment of her existence. And we can easily refute the argument, they claim, by employing perdurantism or exdurantism, according to which a human being is a temporally extended entity with temporal parts or a momentarily existing stage who has other momentarily existing stages as counterparts. I argue that such claims are mistaken. The twinning argument does not rest on endurantism and can be formulated in terms of perdurantism to provide a good reason for perdurantists to reject conceptionism. And exdurantism does not have any advantage in defending conceptionism either, for it already concedes more than what the twinning argument aims to show.     

Comparative growth,cross stress resistance,transcriptomics of Streptococcus pyogenes cultured under low shear modeled microgravity and normal gravity

Streptococcus pyogenes is commonly found on pharynx, mouth and rarely on skin, lower gastrointestinal tract. It is a potential pathogen causing tonsillitis, pneumonia, endocarditis. The present study was undertaken to study the effects of low shear modeled microgravity on growth, morphology, antibiotic resistance, cross-stress resistance to various stresses and alteration in gene expression of S. pyogenes. The growth analysis performed using UV–Visible spectroscopy indicated decrease in growth of S. pyogenes under low shear modeled microgravity. Morphological analysis by Bio-transmission electron microscopy (TEM), Bio-scanning electron microscopy (SEM) did not reveal much difference between normal and low shear modeled microgravity grown S. pyogenes. The sensitivity of S. pyogenes to antibiotics ampicillin, penicillin, streptomycin, kanamycin, hygromycin, rifampicin indicates that the bacterium is resistant to hygromycin. Further S. pyogenes cultured under low shear modeled microgravity was found to be more sensitive to ampicillin and rifampicin as compared with normal gravity grown S. pyogenes. The bacteria were tested for the acid, osmotic, temperature and oxidative cross stress resistances. The gene expression of S. pyogenes under low shear modeled microgravity analyzed by microarray revealed upregulation of 26 genes and down regulation of 22 genes by a fold change of 1.5.     

Measurement of Shoulder Range of Motion in Patients with Adhesive Capsulitis Using a Kinect

Range of motion (ROM) measurements are essential for the evaluation for and diagnosis of adhesive capsulitis of the shoulder (AC). However, taking these measurements using a goniometer is inconvenient and sometimes unreliable. The Kinect (Microsoft, Seattle, WA, USA) is gaining attention as a new motion detecting device that is nonintrusive and easy to implement. This study aimed to apply Kinect to measure shoulder ROM in AC; we evaluated its validity by calculating the agreement of the measurements obtained using Kinect with those obtained using goniometer and assessed its utility for the diagnosis of AC. Both shoulders of 15 healthy volunteers and affected shoulders of 12 patients with AC were included in the study. The passive and active ROM of each were measured with a goniometer for flexion, abduction, and external rotation. Their active shoulder motions for each direction were again captured using Kinect and the ROM values were calculated. The agreement between the two measurements was tested with the intraclass correlation coefficient (ICC). Diagnostic performance using the Kinect ROM was evaluated with Cohen’s kappa value. The cutoff values of the limited ROM were determined in the following ways: the same as passive ROM values, reflecting the mean difference, and based on receiver operating characteristic curves. The ICC for flexion/abduction/external rotation between goniometric passive ROM and the Kinect ROM were 0.906/0.942/0.911, while those between active ROMs and the Kinect ROMs were 0.864/0.932/0.925. Cohen’s kappa values were 0.88, 0.88, and 1.0 with the cutoff values in the order above. Measurements of the shoulder ROM using Kinect show excellent agreement with those taken using a goniometer. These results indicate that the Kinect can be used to measure shoulder ROM and to diagnose AC as an alternative to goniometer.     

Features of replication fork blockage by the Escherichia coli terminus-binding protein.

Blockage of the progress of a DNA replication fork in Escherichia coli can be ascribed to an inhibition of helicase action at the orientation-specific binding of a termination sequence (ter) by the ter-binding protein (Lee, E.H., Kornberg, A., Hidaka, M., Kobayashi, T., and Horiuchi, T. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9104-9108). These observations have been extended to include the PriA helicase, thus confirming that blockage is general for helicases. The site of arrest of synthesis by a replication fork is at the very first nucleotide of the 22-base pair E. coli-terB sequence. Strand displacement by DNA polymerases is also inhibited, but is less profound and is orientation-specific. The ter sequences of plasmids R1-terR and -terL and of plasmids R6K and R100 have been compared with those of E. coli-terA and -terB.