RAD53, DUN1 and PDS1 define two parallel G2/M checkpoint pathways in budding yeast.

Eukaryotic checkpoint genes regulate multiple cellular responses to DNA damage. In this report, we examine the roles of budding yeast genes involved in G2/M arrest and tolerance to UV exposure. A current model posits three gene classes: those encoding proteins acting on damaged DNA (e.g. RAD9 and RAD24), those transducing a signal (MEC1, RAD53 and DUN1) or those participating more directly in arrest (PDS1). Here, we define important features of the pathways subserved by those genes. MEC1, which we find is required for both establishment and maintenance of G2/M arrest, mediates this arrest through two parallel pathways. One pathway requires RAD53 and DUN1 (the 'RAD53 pathway'); the other pathway requires PDS1. Each pathway independently contributes approximately 50% to G2/M arrest, effects demonstrable after cdc13-induced damage or a double-stranded break inflicted by the HO endonuclease. Similarly, both pathways contribute independently to tolerance of UV irradiation. How the parallel pathways might interact ultimately to achieve arrest is not yet understood, but we do provide evidence that neither the RAD53 nor the PDS1 pathway appears to maintain arrest by inhibiting adaptation. Instead, we think it likely that both pathways contribute to establishing and maintaining arrest.     

Polarization of fluorescence from single skinned glycerinated rabbit psoas fibers in rigor and relaxation

Single skinned glycerinated muscle fibers were labelled with the fluorescent dye N-(iodoacetylamino)-1-naphthylamine-5-sulfonic acid (1,5-IAEDANS). The heavy chain of myosin (EC 3.6.1.3) was labelled predominantly when the reaction was carried out in relaxation at 0 °C. Mechanical properties of skinned fibers were little affected by labelling with the fluorophore. Rigor tension developed upon transferring native or labelled skinned fibers from relaxing to rigor solutions lacking Ca²⁺ was very small but could be enhanced by progressively increasing Ca²⁺ concentration; the rigor tension decreased with increasing sarcomere length.Polarization of fluorescence of skinned fibers reacted with 1,5-IAEDANS was measured along the line of excitation as well as at 90° to it. The mean values of parallel and perpendicular components of polarization of labelled fibers measured at 0° were close to the values obtained for native fibers irrigated with 1,5-IAEDANS-labelled heavy meromyosin, fiber “ghosts” irrigated with labelled heavy meromyosin, and oriented bundles of myofibrils reacted with the same fluorophore. Skinned fibers stretched above the rest length and then irrigated with 1,5-IAEDANS-labelled heavy meromyosin gave rise to polarized fluorescence close to the values theoretically predicted for an assembly of helically arranged fluorophores. Using 90° detection system a satisfactory fit to the theory could be obtained from single fibers labelled with 1,5-IAEDANS and measured in rigor. The angle between the fiber axis and the direction of the emission dipole of 1,5-IAEDANS attached to subfragment-1 was estimated to be near 40°.     

Effect of changes of pHi on intracellular calcium in a smooth muscle-like cell line

The effect of changes of pH_i on Ca_i were studied using fluorescent dyes in cells of the cultured smooth muscle-like line, BC3H-1. Resting Ca_i in these cells was 182 ± 12 nM (n = 74) at pH_o of 7.4. Upon exposure to NH₄Cl, which rapidly alkalinized cells, a transient increase of Cai to 349 ± 55 nM (n = 29) was observed. The peak of the transient occurred within 30 s of exposure to NH₄Cl and returned to baseline within 1 minute. Two other procedures which resulted in rapid cellular alkalinization also caused a transient rise in Ca_i: exposure to and then removal of CO₂ (Ca_i increased from 182 ± 22 to 248 ± 28 nM; n = 8); and exposure to and then removal of Na propionate (Ca_i increased from 242 ± 32 to 456 ± 71 nM; n = 9). The NH4Cl-induced Ca_i transient was eliminated by exposure to 0.2 mM TMB8 and to Ca-free solutions, but not by exposure to 0.5 mM LaCl₃. Sustained changes of pH_i can be induced by varying pH_o. When pH_o was lowered to 6.9, Ca_i fell by 49 ± 11 nM but increased by 203 ± 51 nM (n = 6) when pH_o was raised to 7.9. These data indicate that rapid alkalinization of BC3H-1 cells results in a rapid transient rise of Ca_i. This transient is most likely due to the release of Ca from intracellular stores but may also involve an increase of Ca influx. Steady state values of Cai are positively correlated with steady state pH_i. These data may have implications for the contractile state of smooth muscle during periods of acid/base disturbances and relate to the role of elevated pH_i in cells from hypertensive animals.     

Pyrimidine nucleosides enhance the efficacy of inhibitors of pyrimidine biosynthesis in cultured hepatocellular carcinoma cells

Inhibition of colony formation in cultured hepatocellular carcinoma cells of the rat was used to test the efficacy of inhibitors of de novo pyrimidine biosynthesis as potential anticancer drugs. N-(phosphonacetyl)-L-aspartic acid (PALA) (10 and 100 micrograms/ml) and 5-aza-5,6-dihydroorotic acid (DHOX) (100 micrograms/ml) inhibited the formation of colonies and these inhibitions were completely reversed by inclusion of 0.1 mM uridine, the end product of de novo pyrimidine biosynthesis, in the culture medium. With some lots of fetal bovine serum where PALA and DHOX had little effect on inhibiting colony formation, addition of 0.1 mM cytidine restored the inhibitory characteristics of PALA and, to some extent, DHOX. The results demonstrate that cytidine levels modulate the inhibitions of hepatoma colony formation by both PALA and DHOX and that co-administration of these drugs together with cytidine provides a simple expedient to increase drug efficacy.     

Pheromones released during inter- and intra-sex response of the scolytid beetle Dendroctonus brevicomis

Pheromones of Dendroctonus brevicomis released variously during inter- and intra-sex response (including stridulation by both sexes) were the known pheromones exo-brevicomin, endo-brevicomin, frontalin and verbenone, and substances identified as pinocarvone, trans-pinocarveol, and myrtenol. These substances are present in emergent beetles and thus attack of a host tree is not essential for their initial production.     

Self Reported Incidence and Morbidity of Acute Respiratory Illness among Deployed U.S. Military in Iraq and Afghanistan

Background

Historically, respiratory infections have had a significant impact on U.S. military missions. Deployed troops are particularly at high risk due to close living conditions, stressful work environments and increased exposure to pathogens. To date, there are limited data available on acute respiratory illness (ARI) among troops deployed in support of ongoing military operations, specifically Operation Enduring Freedom (OEF) and Operation Iraqi Freedom (OIF).

Methods

Using self-report data from two sources collected from troops deployed to Iraq, Afghanistan and the surrounding region, we analyzed incidence and risk factors for ARI. Military personnel on mid-deployment Rest & Recuperation (R&R) or during redeployment were eligible to participate in the voluntary self-report survey.

Results

Overall, 39.5% reported having at least one ARI. Of these, 18.5% sought medical care and 33.8% reported having decreased job performance. The rate of self-reported ARI was 15 episodes per 100 person-months among those taking the voluntary survey, and 24.7 episodes per 100 person-months among those taking the clinic health questionnaire. Negative binomial regression analysis found female sex, Navy branch of service and lack of flush toilets to be independently associated with increased rates of ARI. Deployment to OIF, increasing age and higher rank were also positively associated with ARI risk.

Conclusions

The overall percentage of deployed military personnel reporting at least one acute respiratory illness decreased since earlier parts of OIF/OEF. However, the reported effect on job performance increased tremendously. The most important factors associated with increased respiratory infection are female sex, Navy branch of service, lack of improved latrine facilities, deployment to OIF, increasing age and higher rank.     

A functionalizable biomaterial based on dihydroxyacetone, an intermediate of glucose metabolism

A biomaterial and its potential degradation products should be biocompatible, nontoxic, and removed by the body upon expiration of its functional lifetime. One historically successful approach is to create new materials from biomolecules that naturally occur in the human body. Herein, we report the synthesis and characterization of a polycarbonate based on dihydroxyacetone, a 3-carbon ketose, and an intermediate in the glucose metabolic pathway. The polymer was synthesized in a range of molecular weights ( approximately 8000 to approximately 37,500) by ring-opening polymerization. The C2 carbonyl of dihydroxyacetone is reactive to amines, and this reactivity was used to functionalize the polymer's surface in a one-step reaction by reductive amination. Additionally, contact angle measurements show the surface of poly(2-oxypropylene carbonate) is hydrophilic even though it is insoluble in water. Mechanical analysis of the polymer revealed it is exceptionally strong for an aliphatic polycarbonate. Specifically, poly(2-oxypropylene carbonate), M(w) 37 500, yielded a Young's modulus of 0.5 GPa and a compressive yield stress of 50 MPa. These values equal or exceed those of cancellous bone with similar dimensions.