Prostaglandin E2 concentrations in naturally occurring canine cancer

The purpose of this study was to determine the PGE2 concentration in naturally-occurring cancer in pet dogs and in canine cancer cell lines in order to identify specific types of canine cancer with high PGE2 production which could serve as preclinical models to evaluate anticancer strategies targeting PGE2. PGE2 concentrations were measured by enzyme immunoassay in canine melanoma, soft tissue sarcoma, transitional cell carcinoma, osteosarcoma, and prostatic carcinoma cell lines; in 80 canine tumor tissue samples including oral melanoma (MEL), oral squamous cell carcinoma (SCC), transitional cell carcinoma of the urinary bladder (TCC), lymphoma (LSA), mammary carcinoma (MCA), osteosarcoma (OSA), prostatic carcinoma (PCA); and in corresponding normal organ tissues. High concentrations of PGE(2)(range 400-3300 pg/10(4)cells) were present in cell culture medium from the transitional cell carcinoma, prostatic carcinoma, and osteosarcoma cell lines. PGE2 concentrations in tumor tissues were elevated (tumor PGE2 concentration>mean+2X sd PGE(2)concentration of normal organ tissue) in 21/22 TCC, 5/6 PCA, 7/10 SCC, 5/10 MEL, 3/8 MCA, 4/15 OSA, and 0/9 LSA. Results of this study will help guide future investigations of anticancer therapies that target cyclooxygenase and PGE2.     

The immunological and metabolic responses to exercise of varying intensities in normoxic and hypoxic environments

The purpose of this study was to determine the effects of varying intensities of exercise in normoxic and hypoxic environments on selected immune regulation and metabolic responses. Using a within-subjects design, subjects performed maximal tests on a cycle ergometer in both normoxic (PiO2 = 20.94%) and hypoxic (PiO2 = 14.65%) environments to determine [latin capital V with dot above]O2max. On separate occasions, subjects then performed four randomly assigned, 1-hour exercise bouts on a cycle ergometer (two each in normoxic and hypoxic environments). The hypoxic environment was created by reducing the O2 concentration of inspired air using a commercially available hypoxic chamber. The intensities for the exercise bouts were predetermined as 40 and 60% of their normoxic [latin capital V with dot above]O2max for the normoxic exercise bouts and as 40 and 60% of their hypoxic [latin capital V with dot above]O2max for the hypoxic exercise bouts. Blood samples were collected preexercise, postexercise, 15 minutes postexercise, 2 hours postexercise, and 24 hours postexercise for the determination of interleukin-1 (IL-1), tumor necrosis factor-[alpha] (TNF-[alpha]), glucose, glycerol, free fatty acids, epinephrine, norepinephrine, and cortisol. There were no significant differences (p < 0.05) between condition or intensity for IL-1 or TNF-[alpha]. Significant differences (p < 0.05) between intensities were demonstrated for epinephrine, norepinephrine, and cortisol (p < 0.05). A significant difference was identified between normoxic and hypoxic environments with respect to nonesterifed fatty acids (0.45 +/- 0.37 vs. 0.58 +/- 0.31 mEq x L-1, respectively; p = 0.012). During prolonged exercise at 40 and 60% of their respective [latin capital V with dot above]O2max values, hypoxia did not seem to dramatically alter the response of the selected immune system or metabolic markers. Exercise training that uses acute hypoxic environments does not adversely affect immune regulation system status and may be beneficial for those individuals looking to increase endurance performance.     

Antenna to leg transformation: Dynamics of developmental competence

We explore the transformation of antenna to leg in Drosophila melanogaster, using ectopically expressed transgenes with heat shock promoters: heat shock Antennapedia, heat shock Ultrabithorax, and heat shock mouse Hox A5. We determined the frequency of transformation of several leg markers in response to Antennapedia protein delivered by heat shock at different times and doses. We also studied stage-specific responses to the transgene, heat shock mouse Hox A5. Results show that each marker has its own stage and dose-specific pattern of response. The same marker could pass through a period of high-dose inhibition followed by a dose-independent response and then a positive dose-dependent phase. The heat shock-induced transgenes and spineless aristapedia transformed the apterous enhancer trap antenna disc expression pattern toward the pattern found in leg discs. These results are considered in relation to developmental competence—the ability of developing tissue to respond to internal or external influences. The results suggest that all genes tested interact with the same competence system and that at least two classes of mechanisms are associated with antenna to leg transformation: one comprises global mechanisms that permit transformation over approximately 24 hr; the second class of mechanisms act very locally and are responsible for changes in dose response on the order of 4–8 hr. © 1996 Wiley-Liss, Inc.     

Biochemical and genetic analysis of the four DNA ligases of mycobacteria

Mycobacterium tuberculosis encodes an NAD(+)-dependent DNA ligase (LigA) plus three distinct ATP-dependent ligase homologs (LigB, LigC, and LigD). Here we purify and characterize the multiple DNA ligase enzymes of mycobacteria and probe genetically whether the ATP-dependent ligases are required for growth of M. tuberculosis. We find significant differences in the reactivity of mycobacterial ligases with a nicked DNA substrate, whereby LigA and LigB display vigorous nick sealing activity in the presence of NAD(+) and ATP, respectively, whereas LigC and LigD, which have ATP-specific adenylyltransferase activity, display weak nick joining activity and generate high levels of the DNA-adenylate intermediate. All four of the mycobacterial ligases are monomeric enzymes. LigA has a low K(m) for NAD(+) (1 microm) and is sensitive to a recently described pyridochromanone inhibitor of NAD(+)-dependent ligases. LigA is able to sustain growth of Saccharomyces cerevisiae in lieu of the essential yeast ligase Cdc9, but LigB, LigC, and LigD are not. LigB is distinguished by its relatively high K(m) for ATP (0.34 mm) and its dependence on a distinctive N-terminal domain for nick joining. None of the three ATP-dependent ligases are essential for mycobacterial growth. M. tuberculosis ligDDelta cells are defective in nonhomologous DNA end joining.     

DNA Ligase C1 Mediates the LigD-Independent Nonhomologous End-Joining Pathway of Mycobacterium smegmatis

Nonhomologous end joining (NHEJ) is a recently described bacterial DNA double-strand break (DSB) repair pathway that has been best characterized for mycobacteria. NHEJ can religate transformed linear plasmids, repair ionizing radiation (IR)-induced DSBs in nonreplicating cells, and seal I-SceI-induced chromosomal DSBs. The core components of the mycobacterial NHEJ machinery are the DNA end binding protein Ku and the polyfunctional DNA ligase LigD. LigD has three autonomous enzymatic modules: ATP-dependent DNA ligase (LIG), DNA/RNA polymerase (POL), and 3′ phosphoesterase (PE). Although genetic ablation of ku or ligD abolishes NHEJ and sensitizes nonreplicating cells to ionizing radiation, selective ablation of the ligase activity of LigD in vivo only mildly impairs NHEJ of linearized plasmids, indicating that an additional DNA ligase can support NHEJ. Additionally, the in vivo role of the POL and PE domains in NHEJ is unclear. Here we define a LigD ligase-independent NHEJ pathway in Mycobacterium smegmatis that requires the ATP-dependent DNA ligase LigC1 and the POL domain of LigD. Mycobacterium tuberculosis LigC can also support this backup NHEJ pathway. We also demonstrate that, although dispensable for efficient plasmid NHEJ, the activities of the POL and PE domains are required for repair of IR-induced DSBs in nonreplicating cells. These findings define the genetic requirements for a LigD-independent NHEJ pathway in mycobacteria and demonstrate that all enzymatic functions of the LigD protein participate in NHEJ in vivo.     

Getting in and out of the proteasome

By far the best understood role of the proteasome is to remove ubiquitin-conjugated proteins from eukaryotric cells by hydrolysing them into small peptides of varying lengths. These include both misfolded/abnormal proteins, as well as 'normal' proteins that need to be rapidly removed for regulatory purposes. However, the proteasome is also present in numerous prokaryotic organisms, while ubiquitin, and the ubiquitin conjugating system, are not. The eukaryotic proteasome has been adapted to degrading proteins in a ubiquitin-dependent fashion by the addition of regulatory factors that assemble in different layers onto the proteolytic core of the proteasome, and by increasing the diversity of the core subunits as well. In addition to hydrolysing ubiquitinated proteins into amino acids, the proteasome can also proteolyse selected non-ubiquitinated proteins, process proteins, and possibly refold misfolded proteins. This review will focus on the different proteasome functions, and how these are used in the multiple regulatory roles the proteasome plays in eukaryotic cells.     

An adaptable HPLC method for the analysis of frequently used antibiotics in ocular samples

Four different antibiotics, delivered individually to rabbit eyes via hydrophilic intraocular lenses soaked in the drug solution prior to implantation, were measured in aqueous and vitreous humor samples from the eyes. To meet this analytical need, we developed a sensitive, high performance liquid chromatographic (HPLC) method for measuring the concentrations of moxifloxacin, gatifloxacin, linezolid, and cefuroxime in the ocular tissue. Separations were carried out on a LichroSpher RP-18 column, maintained at room temperature. The fluoroquinolones were eluted with a mobile phase consisting of 20% acetonitrile, in 0.1% trifluoroacetic acid (pH 3.0) with 30 mM tetrabutylammonium chloride. Linezolid and cefuroxime were eluted with 25% acetonitrile in 25 mM Na acetate buffer, pH 5.0. All elutions were isocratic. With ultraviolet detection, the lower limit of quantitation (LLOQ) for these compounds approached 1 ng (on-column injection). By using fluorescence detection, the LLOQ for the fluoroquinolones improved to 200 pg. The overall accuracy of the method was ≥90%. With minor modifications, the method was optimized for each of the agents, and the resulting analytical sensitivity made the method suitable for clinical investigations of the ocular penetration of these drugs.