Identification of two major direct repeat unit clusters, 8i and 11ce,among methicillin resistant Staphylococcus aureus strains: the emergence of novel dru types and repeats

Molecular Biology Reports - The changing epidemiology and decreasing susceptibility to first-line antibiotics, such as vancomycin and linezolid, leave clinicians with few therapeutic options for...     

Evaluation of chemical, physical, and biologic properties of tumor-targeting radioiodinated quinazolinone derivative

Our group is developing a novel technology, enzyme-mediated cancer imaging and therapy (EMCIT), that aims to entrap radioiodinated compounds within solid tumors for noninvasive tumor detection and therapy. In this approach, a water-soluble, radioiodinated prodrug is hydrolyzed in vivo to a highly water-insoluble compound by an enzyme overexpressed extracellularly by tumor cells. We have synthesized and characterized the water-soluble prodrug, 2-(2'-phosphoryloxyphenyl)-6-[(125)I]iodo-4-(3H)-quinazolinone [(125)I]5, which is readily hydrolyzed by alkaline phosphatase, an enzyme expressed by many tumor cell lines, to a water-insoluble drug, 2-(2'-hydroxyphenyl)-6-[(125)I]iodo-4-(3H)-quinazolinone [(125)I]1. In the course of our study, we discovered that ammonium 2-(2'-phosphoryloxyphenyl)-6-tributylstannyl-4-(3H)-quinazolinone, an intermediate in the radioiodination of the prodrug, exists as two isomers (3 and 4) whose radioiodination leads, respectively, to [(125)I]6 and [(125)I]5. These prodrugs have different in vitro and in vivo biologic activities. Compound 6 is not hydrolyzed by alkaline phosphatase (ALP), whereas 5 is highly soluble (mg/mL) in aqueous solution and is rapidly dephosphorylated in the presence of ALP to 1, a water-insoluble molecule (ng/mL). Mouse biodistribution studies indicate that [(125)I]6 has high uptake in kidney and liver and [(125)I]5 has very low uptake in all normal organs. Compounds 3 and 6 are converted, respectively, to 4 and 5 after incubation in DMSO. The stability of 5 in human serum is high. The minimum ALP concentration needed to hydrolyze 5 is much greater than the ALP level in the blood of patients with cancer, and the latter should not affect the pharmacokinetics of the compound. Incubation of 5 with viable human and mouse tumor-cell lines--but not with normal human cells and mouse tissues--leads to its hydrolysis and the formation of large crystals of 1. We expect that 5 will also be hydrolyzed in vivo by tumor cells that express phosphatase activity extracellularly and anticipate the specific precipitation of radioiodinated 1 within tumor cell clusters. This should lead to high tumor-to-normal-tissue ratios and enable imaging (SPECT/PET) and radionuclide therapy of solid tumors.     

Cdk5 Modulation of mitogen-activated protein kinase signaling regulates neuronal survival

Cdk5, a cyclin-dependent kinase, is critical for neuronal development, neuronal migration, cortical lamination, and survival. Its survival role is based, in part, on "cross-talk" interactions with apoptotic and survival signaling pathways. Previously, we showed that Cdk5 phosphorylation of mitogen-activated protein kinase kinase (MEK)1 inhibits transient activation induced by nerve growth factor (NGF) in PC12 cells. To further explore the nature of this inhibition, we studied the kinetics of NGF activation of extracellular signal-regulated kinase (Erk)1/2 in cortical neurons with or without roscovitine, an inhibitor of Cdk5. NGF alone induced an Erk1/2-transient activation that peaked in 15 min and declined rapidly to baseline. Roscovitine, alone or with NGF, reached peak Erk1/2 activation in 30 min that was sustained for 48 h. Moreover, the sustained Erk1/2 activation induced apoptosis in cortical neurons. Significantly, pharmacological application of the MEK1 inhibitor PD98095 to roscovitine-treated cortical neurons prevented apoptosis. These results were also confirmed by knocking down Cdk5 activity in cortical neurons with Cdk5 small interference RNA. Apoptosis was correlated with a significant shift of phosphorylated tau and neurofilaments from axons to neuronal cell bodies. These results suggest that survival of cortical neurons is also dependent on tight Cdk5 modulation of the mitogen-activated protein kinase signaling pathway.     

Expression of the neural stem cell markers NG2 and L1 in human angiomyolipoma: are angiomyolipomas neoplasms of stem cells?

Angiomyolipomas are benign tumors of the kidney which express phenotypes of smooth muscle, fat, and melanocytes. These tumors appear with increased frequency in the autosomal dominant disorder tuberous sclerosis and are the leading cause of morbidity in adults with tuberous sclerosis. While benign, these tumors are capable of provoking life threatening hemorrhage and replacement of the kidney parenchyma, resulting in renal failure. The histogenesis of these tumors is currently unclear, although currently, we believe these tumors arise from "perivascular epithelioid cells" of which no normal counterpart has been convincingly demonstrated. Recently, stem cell precursors have been recognized that can give rise to smooth muscle and melanocytes. These precursors have been shown to express the neural stem cell marker NG2 and L1. In order to determine whether angiomyolipomas, which exhibit smooth muscle and melanocytic phenotypes, express NG2 and L1, we performed immunocytochemistry on a cell line derived from a human angiomyolipoma, and found that these cells are uniformly positive. Immunohistochemistry of human angiomyolipoma specimens revealed uniform staining of tumor cells, while renal cell carcinomas revealed positivity only of angiogenic vessels. These results support a novel histogenesis of angiomyolipoma as a defect in differentiation of stem cell precursors.     

Fractalkine-induced MFG-E8 leads to enhanced apoptotic cell clearance by macrophages

Clearance of apoptotic cells is crucial to maintain cellular function under normal and pathological conditions. We have recently shown that administration of immature dendritic cell-derived exosomes to septic animals promotes phagocytosis of apoptotic cells and improves survival by providing milk fat globule epidermal growth factor-factor VIII (MFG-E8). MFG-E8 acts as an opsonin for apoptotic cells to be engulfed by phagocytosis. In the present study we investigated whether the CX(3)C-chemokine fractalkine (CX(3)CL1) promotes apoptotic cell clearance through the induction of MFG-E8 in peritoneal macrophages. Cultured rat peritoneal macrophages (pMphi) and RAW264.7 macrophages were stimulated with LPS and CX(3)CL1. MFG-E8 expression was assessed by Western blot, cytokine secretion was assessed by ELISA, and phagocytosis of apoptotic thymocytes was determined by microscopy. For in vivo studies, cecal ligation and puncture (CLP) was used to induce sepsis in rats and mice. LPS significantly decreased MFG-E8 levels and phagocytosis of apoptotic cells, whereas CX(3)CL1 induced MFG-E8 expression in both nonstimulated and LPS-stimulated pMphi, without affecting TNF-alpha and IL-6 release. Anti-MFG-E8 blocking antibodies completely abrogated the prophagocytic effect of CX(3)CL1. Twenty hours after the induction of sepsis in rats via CLP, plasma CX(3)CL1 levels as well as MFG-E8 production in peritoneal macrophages decreased by 21% and 56%, respectively. Administration of CX(3)CL1 on the other hand induced MFG-E8 and prevented tissue injury. We conclude that CX(3)CL1 induces MFG-E8 in vitro and in vivo and enhances clearance of apoptotic cells in an MFG-E8-dependent manner. These findings suggest a possible novel treatment for patients in sepsis.     

The expression and genetics of resistance to stripe (yellow) rust in three European and four New Zealand wheat cultivars

Adult plant resistance (APR) to stripe rust in three European (Pegaso, Victo and Aztec) and four New Zealand cultivars (Weka, Kopara, Kokart and Takahe) was characterised using hybrid analysis and tests of allelism. In agreement with earlier work, the APR in most of these cultivars appeared to be controlled by two or more genes with additive effects. It was suggested that heavy selection pressure should be avoided in early generations in breeding programs utilising APR, because lines in which APR genes are heterozygous may display lower levels of resistance due to the incompletely dominant and interactive nature of many APRs. Such lines are capable of generating more resistant progenies following selfing. It was also demonstrated that it is possible to misclassify F2 plants as susceptible if APR genes are in a heterozygous condition, especially in the case of gene(s) conferring intermediate levels of resistance. The presence of a common APR gene in Kopara and Takahe, and perhaps Weka, was suggested because all shared a common parent in their pedigree and no susceptible plants were observed in F2 populations derived from intercrossing them. The difficulties inherent in conducting genetic studies on APRs and the need for large population sizes for such studies were emphasised.     

Effects of 1,4-phenylenebis(methylene)selenocyanate on mutagenesis and p53 protein expression in the tongue of lacI rats treated with 4-nitroquinoline-N-oxide

Previously we showed that the organoselenium compound, 1,4-phenylenebis(methylene)selenocyanate (p-XSC)(1) inhibits 4-nitroquinoline-N-oxide (4-NQO)-induced tongue tumorigenesis in Fisher rats. Here we investigate possible mechanisms of this inhibition by monitoring mutagenesis and p53 protein levels in lacI and conventional Fisher rats treated with: (1) a carcinogenic dose of 4-NQO for 10 weeks in drinking water, (2) 4-NQO+p-XSC (15 ppm as selenium), and (3) 4-NQO followed by p-XSC. For mutagenesis studies, rats were euthanized at 7, 12 or 23 weeks after the start of 4-NQO. For studies on p53 levels, rats were euthanized at 11, 15 and 23 weeks. Appropriate controls were also monitored. In the 4-NQO-alone groups, the mutant fraction (MF) in the cII gene in tongue increased at least 50x background level. The MF (in units of mutants/10(5) plaque forming units) for the 7, 12, and 23 weeks 4-NQO groups were respectively, 184 +/- 88, 237 +/- 105, and 329 +/- 110. Thus, mutagenesis increased with length of exposure and post-treatment time. p-XSC modestly (ca. 15-30%) inhibited mutagenesis under all conditions. The inhibition reached significance at the last time point. When p-XSC was administered after 4-NQO, the MF was also modestly reduced. In 4-NQO-alone animals, levels of p53 in tongue (determined by Western blotting) were 1, 1.5 and 2.4 control levels at 10, 15 and 23 weeks, respectively. In the p-XSC+4-NQO group, the enhancement in p53 levels by 4-NQO treatment was decreased about 90% at 15 weeks and 45% (P<0.05) at 23 weeks, and by slightly smaller percentages in corresponding post-treatment groups. p-XSC alone did not alter p53 levels. As p53 levels generally increase in response to DNA damage, these results suggest that p-XSC reduces 4-NQO-induced DNA damage, resulting in reduced 4-NQO-induced mutagenesis and carcinogenesis. However, the fact that p-XSC is also effective when administered after 4-NQO, suggests additional mechanisms of inhibition exist.     

Glucose regulation of pre-steady state kinetics of ATP hydrolysis by Na,K-ATPase

The effect of glucose and 2-deoxy-D-glucose on pre-steady state kinetics of ATP hydrolysis by Na,K-ATPase has been investigated by following pH transients in a stopped-flow spectrophotometer. A typical pre-steady state signal showed an initial decrease then subsequent increase in acidity. Under optimal Na^＋ （120 mM） and K^＋ （30 mM） concentrations, magnitudes of both H^＋ release and H^＋ absorption were found to be approximately 1.0/ATPase molecule. The presence of 1 mM glucose significantly decreased H^＋ absorption at high Na^＋ concentrations, whereas it was ineffective at low Na^＋. H^＋ release was decreased significantly in the presence of 1 mM glucose at Na^＋ concentrations ranging from 30 mM to 120 mM. Similar to the control, K^＋ did not show any effect on either H^＋ release or H^＋ absorption at all tested combinations of Na^＋ and K^＋ concentrations. Pre-steady state H^＋ signal obtained in the presence of 2-deoxy-D-glucose did not vary significantly as compared with glucose. Delayed addition of K^＋ （by 30 ms） to the mixture （enzyme＋ 120 mM Na^＋ATP＋glucose） showed that only small fractions of population absorb H^＋ in the absence of K^＋. No H^＋ absorption was observed in the absence of Na^＋. Delayed mixing of Na^＋ or K^＋ did not have any effect on H^＋ release. Effect of 2-deoxy-D-glucose on H^ absorption and release was almost the same as that of glucose at all combinations of Na^＋ and K^＋ concentrations. Results obtained have been discussed in terms of an extended kinetic scheme which shows that, in the presence of either glucose or 2-deoxy-D-glucose, significantly fewer enzyme molecules reache the E-P（3Na＋） stage and that K^ plays an important role in the conversion of E1 .ADP.P（3Na^＋） to H^＋.E1-（3Na^＋） complex.     

Process optimization and characterization of poloxamer solid dispersions of a poorly water-soluble drug

The objective of the present investigation was to improve the dissolution rate of Rofecoxib (RXB), a poorly water-soluble drug by solid dispersion technique using a water-soluble carrier, Poloxamer 188 (PXM). The melting method was used to prepare solid dispersions. A 3² full factorial design approach was used for optimization wherein the temperature to which the melt-drug mixture cooled (X ₁) and the drug-to-polymer ratio (X ₂) were selected as independent variables and the time required for 90% drug dissolution (t₉₀) was selected as the dependent variable. Multiple linear regression analysis revealed that for obtaining higher dissolution of RXB from PXM solid dispersions, a low level ofX ₁ and a high level ofX ₂ were suitable. The differential scanning calorimetry and x-ray diffraction studies demonstrated that enhanced dissolution of RXB from solid dispersion might be due to a decrease in the crystallinity of RXB and PXM and dissolution of RXB in molten PXM during solid dispersion preparation. In conclusion, dissolution enhancement of RXB was obtained by preparing its solid dispersions in PXM using melting technique. The use of a factorial design approach helped in identifying the critical factors in the preparation and formulation of solid dispersion. Published: April 13, 2007