Handoff from recombinase to replisome: Insights from transposition

Bacteriophage Mu replicates as a transposable element, exploiting host enzymes to promote initiation of DNA synthesis. The phage-encoded transposase MuA, assembled into an oligomeric transpososome, promotes transfer of Mu ends to target DNA, creating a fork at each end, and then remains tightly bound to both forks. In the transition to DNA synthesis, the molecular chaperone ClpX acts first to weaken the transpososome's interaction with DNA, apparently activating its function as a molecular matchmaker. This activated transpososome promotes formation of a new nucleoprotein complex (prereplisome) by yet unidentified host factors [Mu replication factors (MRF alpha 2)], which displace the transpososome in an ATP-dependent reaction. Primosome assembly proteins PriA, PriB, DnaT, and the DnaB--DnaC complex then promote the binding of the replicative helicase DnaB on the lagging strand template of the Mu fork. PriA helicase plays an important role in opening the DNA duplex for DnaB binding, which leads to assembly of DNA polymerase III holoenzyme to form the replisome. The MRF alpha 2 transition factors, assembled into a prereplisome, not only protect the fork from action by nonspecific host enzymes but also appear to aid in replisome assembly by helping to activate PriA's helicase activity. They consist of at least two separable components, one heat stable and the other heat labile. Although the MRF alpha 2 components are apparently not encoded by currently known homologous recombination genes such as recA, recF, recO, and recR, they may fulfill an important function in assembling replisomes on arrested replication forks and products of homologous strand exchange.     

Thrombomodulin activity is found in tissue thromboplastin preparations from placenta and from lung but not from brain

Substantial thrombomodulin activity could be detected in tissue thromboplastin preparations from placenta or from lung but not from brain. When the amount of these preparations was adjusted to contain 1 unit of tissue factor activity, up to 0.85 units of thrombomodulin activity could be measured, corresponding to the generation of 17 pmol/ml/min of activated protein C when 1.5 microM human protein C was activated by 20 nM human alpha-thrombin in the presence of 5 mM CaCl2. After treatment by phospholipase C, thrombomodulin activity was reduced in these samples. Addition of mixed brain procoagulant phospholipids partially restored thrombomodulin activity in the phospholipase C-treated samples. These results emphasize the role of phospholipids in the expression of optimal thrombomodulin activity in tissue thromboplastin preparations from placenta or from lung.     

Hemoglobin loss from erythrocytes in vivo results from spleen-facilitated vesiculation

Previous studies have shown that approximately 20% of hemoglobin is lost from circulating red blood cells (RBCs), mainly during the second half of the cells' life span. Because hemoglobin-containing vesicles are known to circulate in plasma, these vesicles were isolated. Flow cytometry studies showed that most RBC-derived vesicles contain hemoglobin with all hemoglobin components present. The hemoglobin composition of the vesicles resembled that of old RBCs. RBC cohort studies using isotope-labeled glycine have been described, which showed a continuous presence of this label in hemoglobin degradation products. The label concentration of these products increased during the second half of the RBC life span, accompanied by a decrease within the RBC. It is concluded that the hemoglobin loss from circulating RBCs of all ages can be explained by shedding hemoglobin-containing vesicles. This loss occurs predominantly in older RBCs. Apparently the spleen facilitates this process since asplenia vesicle retention within RBCs of all ages has been described, accompanied by an increase in the percentage of total HbA(1). The present study shows that in old RBCs of asplenic individuals, the decrease of hemoglobin content per cell such as seen in old RBCs of control individuals is absent due to an increase in the absolute amount of HbA(1c) and HbA(1e2). It is concluded that hemoglobin-containing vesicles within old RBCs are "pitted" by the spleen.     

Learning More from Microarrays: Insights from Modules and Networks

Global gene expression patterns can provide comprehensive molecular portraits of biologic diversity and complex disease states, but understanding the physiologic meaning and genetic basis of the myriad gene expression changes have been a challenge. Several new analytic strategies have now been developed to improve the interpretation of microarray data. Because genes work together in groups to carry out specific functions, defining the unit of analysis by coherent changes in biologically meaningful sets of genes, termed modules, improves our understanding of the biological processes underlying the gene expression changes. The gene module approach has been used in exploratory discovery of defective oxidative phosphorylation in diabetes mellitus and also has allowed definitive hypothesis testing on a genomic scale for the relationship between wound healing and cancer and for the oncogenic mechanism of cyclin D. To understand the genetic basis of global gene expression patterns, computational modeling of regulatory networks can highlight key regulators of the gene expression changes, and many of these predictions can now be experimentally validated using global chromatin-immunoprecipitation analysis.     

Isolation of Circular DNA from a Mitochondrial Fraction from Yeast

Breakage and fractionation of respiratory competent yeast in the presence of ethidium bromide, and subsequent centrifugation of a detergent lysate of the mitochondrial fraction by the dye-buoyant-density technique, results in the isolation of closed-circular DNA. After removal of bound dye, this DNA has two components when analyzed by equilibrium buoyant density in the analytical ultracentrifuge. A minor component has a buoyant density of 1.684 g/cm(3), which is characteristic of mitochondrial DNA, but the major component has a buoyant density of 1.701 g/cm(3). This species of DNA is also present in yeast that have been mutagenized to respiratory deficiency in the presence of the highest concentration of ethidium bromide compatible with cell growth. The closed-circular DNA of buoyant density 1.701 g/cm(3), and free of linear DNA, is associated with the sole particulate band obtained on sucrose gradient centrifugation of a mitochondrial preparation from respiratory-deficient cells. Two particulate bands are obtained on sucrose gradient centrifugation of a mitochondrial preparation from respiratory-competent cells, the upper band containing DNA of buoyant density 1.701 g/cm(3) and the lower band DNA of buoyant density 1.684 g/cm(3). The suggestion is advanced, in view of the reputed sedimentation behaviour of yeast peroxisomes, that the closed-circular DNA of buoyant density 1.701 g/cm(3) may be located in peroxisomes.     

RNA-directed RNA polymerases from healthy and from virus-infected cucumber.

Much work has been done on the isolation, purification, and characterization of the RNA-directed RNA polymerase (EC 2.7.7.48) of cucumber mosaic virus (CMV)-infected cucumbers. Uninfected plants were reported to have no such enzyme, but we recently detected low levels of the activity in cucumber. Since tobacco and cowpea contain such an enzyme that is variably increased in amount by various virus (as well as viroid) infections, we assumed that this would also be the case upon CMV infection of cucumber. However, further purification and characterization of the RNA-directed RNA polymerases from healthy and from infected cucumber suggests that these are different enzymes. The presumed CMV replicase was obtained pure and consists of a major polypeptide of Mr 100,000 and minor components of Mr 110,000 and about 10,000. The Km is 5 microM ([3H]GTP) when tobacco mosaic virus RNA is used as template.     

Isolation of gram-negative bacteria from cockroaches trapped from urban environment

Three different areas--hospital, food-handling establishments and human dwellings, were surveyed for pathogenic gram-negative bacteria carried on the cuticles of cockroaches. Fifty species of bacteria were identified from all cockroaches. Escherichia coli, Klebsiella pneumoniae, Citrobacter freundii and Enterobacter cloacae were the most frequently found. Pathogenic and potentially pathogenic bacteria represented 58% of all bacteria identified. The numbers of pathogenic and potentially pathogenic bacteria were similar in hospital areas and food-handling establishments, while, human dwellings possessed a poorer bacterial flora. E. coli, K. pneumoniae and E. cloacae were dominant species in hospital areas, while in food-handling establishments and human dwellings, E. coli, K. pneumoniae and C. freundii predominated. Therefore, cockroaches can play a role in bacterial transmission, due to the bacteria carried on their cuticles.     

Lessons from the past: biotic recoveries from mass extinctions

Although mass extinctions probably account for the disappearance of less than 5% of all extinct species, the evolutionary opportunities they have created have had a disproportionate effect on the history of life. Theoretical considerations and simulations have suggested that the empty niches created by a mass extinction should refill rapidly after extinction ameliorates. Under logistic models, this biotic rebound should be exponential, slowing as the environmental carrying capacity is approached. Empirical studies reveal a more complex dynamic, including positive feedback and an exponential growth phase during recoveries. Far from a model of refilling ecospace, mass extinctions appear to cause a collapse of ecospace, which must be rebuilt during recovery. Other generalities include the absence of a clear correlation between the magnitude of extinction and the pace of recovery or the resulting ecological and evolutionary disruption the presence of a survival interval, with few originations, immediately after an extinction and preceding the recovery phase, and the presence of many lineages that persist through an extinction event only to disappear during the subsequent recovery. Several recoveries include numerous missing lineages, groups that are found before the extinction, then latter in the recovery, but are missing during the initial survival-recovery phase. The limited biogeographic studies of recoveries suggest considerable variability between regions.     

Crop pollination from native bees at risk from agricultural intensification

Ecosystem services are critical to human survival; in selected cases, maintaining these services provides a powerful argument for conserving biodiversity. Yet, the ecological and economic underpinnings of most services are poorly understood, impeding their conservation and management. For centuries, farmers have imported colonies of European honey bees (Apis mellifera) to fields and orchards for pollination services. These colonies are becoming increasingly scarce, however, because of diseases, pesticides, and other impacts. Native bee communities also provide pollination services, but the amount they provide and how this varies with land management practices are unknown. Here, we document the individual species and aggregate community contributions of native bees to crop pollination, on farms that varied both in their proximity to natural habitat and management type (organic versus conventional). On organic farms near natural habitat, we found that native bee communities could provide full pollination services even for a crop with heavy pollination requirements (e.g., watermelon, Citrullus lanatus), without the intervention of managed honey bees. All other farms, however, experienced greatly reduced diversity and abundance of native bees, resulting in insufficient pollination services from native bees alone. We found that diversity was essential for sustaining the service, because of year-to-year variation in community composition. Continued degradation of the agro-natural landscape will destroy this "free" service, but conservation and restoration of bee habitat are potentially viable economic alternatives for reducing dependence on managed honey bees.     

Learning from health system reforms: lessons from Burkina Faso

OBJECTIVES: Burkina Faso has implemented a macroeconomic adjustment programme (MAP) along with an ambitious reform of the health care system. Our aim was (1) to verify whether MAPs led to a reduction in health resources, and (2) to analyze the consequences of health policies implemented. METHOD: Cross-sectional and retrospective study, spanning the years 1983-2003. The macro aspect is based upon documents from national and international sources, a database of secondary socioeconomic data, and interviews of key informants working in upper management. Household and health facility surveys were conducted in three regions covering 53 communities. RESULTS: Within the reforms, the health sector benefited from an important flow of resources. There were significant increases in public expenditures, health care staff, the number of primary care facilities and the availability of generic drugs. However, health facilities in the public sector remain underused and major inequities subsist. Access to health care is constrained by the population's ability to pay. Health expenditures impoverish households, creating new poor and impoverishing the already poor. CONCLUSIONS: The success of reforms depends largely on the extent to which they remove financial barriers to access to services. The experience of Burkina Faso also reveals the need for fundamental changes that will motivate staff, improve productivity, and ensure good quality services. Integrating health development policies with strategic plans for poverty reduction can provide new opportunities for African countries to redesign their health systems within this type of perspective.     

Methane emissions from Alaska in 2012 from CARVE airborne observations

We determined methane (CH₄) emissions from Alaska using airborne measurements from the Carbon Arctic Reservoirs Vulnerability Experiment (CARVE). Atmospheric sampling was conducted between May and September 2012 and analyzed using a customized version of the polar weather research and forecast model linked to a Lagrangian particle dispersion model (stochastic time-inverted Lagrangian transport model). We estimated growing season CH₄ fluxes of 8 ± 2 mg CH₄⋅m⁻²⋅d⁻¹ averaged over all of Alaska, corresponding to fluxes from wetlands of 56−13+22 mg CH₄⋅m⁻²⋅d⁻¹ if we assumed that wetlands are the only source from the land surface (all uncertainties are 95% confidence intervals from a bootstrapping analysis). Fluxes roughly doubled from May to July, then decreased gradually in August and September. Integrated emissions totaled 2.1 ± 0.5 Tg CH₄ for Alaska from May to September 2012, close to the average (2.3; a range of 0.7 to 6 Tg CH₄) predicted by various land surface models and inversion analyses for the growing season. Methane emissions from boreal Alaska were larger than from the North Slope; the monthly regional flux estimates showed no evidence of enhanced emissions during early spring or late fall, although these bursts may be more localized in time and space than can be detected by our analysis. These results provide an important baseline to which future studies can be compared.Recent studies have raised concerns about an increase in methane (CH₄) emissions from Arctic regions as temperatures warm (1–3). Carbon stocks in polar regions are estimated to be as large as 1,700 Pg of soil organic carbon (4), preserved by cold, wet conditions that inhibit decomposition. Over the last 20 y, temperatures have increased more rapidly at these latitudes than the rest of the world (5); continuation of this trend will lead to permafrost warming and thawing (6), potentially releasing vast quantities of carbon dioxide (CO₂) and CH₄ into the atmosphere (7–10). A recent synthesis of carbon emissions predicted by permafrost models reported releases in the range of 120 ± 85 Pg C by 2100 (11). Large uncertainties are likewise associated with estimates of CH₄ emissions (12–90 Tg CH₄⋅y⁻¹) (12). The potential for large increases in CH₄ emissions are a particular concern because CH₄ strongly impacts both atmospheric chemistry and climate (13). Estimates of the impact of permafrost carbon emissions on future global temperatures range from ∼0.1–0.2 °C (14) to 0.3 ± 0.2 °C (11) by 2100, with increased carbon emissions expected to continue after 2100 (11).Recent global inversion studies find no evidence for increasing CH₄ emissions from these regions in the last 10 y (15, 16), despite warming, similar to earlier studies (17–19) and some biogeochemical models (14). Surface CH₄ flux observations across the pan-Arctic from 1990–2006 have ranged widely and measurement locations have changed, making it difficult to detect any trend over those years (ref. 20; cf. ref. 21).The present paper derives estimates of CH₄ surface fluxes in Alaska from May to September 2012, based on an extensive program of regional-scale airborne measurements of atmospheric CH₄, the Carbon in Arctic Reservoirs Vulnerability Experiment (CARVE). We quantify the monthly mean CH₄ emissions from Alaska during the growing season, providing a snapshot of the interactions between climate and the vast reservoir of preserved soil organic matter in the Arctic.     

Haemagglutinins from Salvia

Abstract. Agglutinins from the seeds of various Salvia species were studied. Useful Tn-specific agglutinins can be prepared from Salvia sclarea and Salvia haematodes. Extracts of the seeds of Salvia farinacea and Salvia horminum contain separable anti-Tn and anti-Cad agglutinins. These agglutinins are of potential value in the study of erythrocyte polyagglutination.     

News from UKPDS

Risk factors present in type 2 diabetic patients at the time of their inclusion in the study were related with the occurrence of some complications. Thus, relative risk for coronary heart disease is 1.57 when HbA1c is > 7.5%, 1.41 when LDL-Cholesterol is > 3.89 mmol/l, these factors are not involved in the occurrence of cerebral stroke. Blood pressure and HbA1c were monitored throughout the study and could have been significantly correlated with the occurrence of micro- and macroangiopathy, with a linear-type and even exponential (HbA1c/retinopathy) relationship and a synergistic interaction between these 2 parameters. A treatment allowing a 0.9% decrease of HbA1c and a 10 mmHg decrease of systolic blood pressure is associated with a reduction of complications, more or less important than that expected from the epidemiological analysis of data. Several therapeutic strategies ensue from these observations.     

Lessons from ONTARGET

The recently published results of the ONTARGET trial shed a new light on the cardiovascular protection of patients at high risk of a cardiovascular event. Despite a number of trials looking at the efficacy of Angiotensin Converting Enzyme inhibitors (ACEis) or Angiotensin Receptor Blockers (ARBs) in the prevention of cardiovascular events in patients with specific high risk profiles, the question of the equivalence of ACEis and ARBs remained unanswered. The ONTARGET trial has shown that telmisartan 80 mg administered for a median duration of 4.5 years to patients at high risk of developing a major cardiovascular event, is equally effective to ramipril 10 mg. In addition, telmisartan was slightly better tolerated. The comparator ramipril has been chosen as it is currently the gold standard ACEi since the results of the HOPE study, in terms of the composite outcome of cardiovascular death, myocardial infarction and stroke. Moreover, ONTARGET is the first trial to test the hypothesis of superiority of adding an ARB (telmisartan 80 mg) to an ACEi (ramipril 10 mg) over the ACEi ramipril monotherapy in cardiovascular protection of the same broad range of high-risk patients. Surprisingly, despite a more pronounced blood pressure lowering, the combination of the two agents did not lead to an additional decrease in the number of events, but had significantly more side-effects compared to ramipril monotherapy. ONTARGET is a landmark study, performed according to the highest statistical and clinical standards, providing compelling evidence and clear answers to two important clinical questions.     

Lessons from Eurodiale

The European Study Group on Diabetes and the Lower Extremity is a collaborative, multi-disciplinary network of 14 centres from ten countries. In 2003-2004, all patients treated in the participating centres because of a new diabetic foot ulcer (n=1232) were included in a prospective observational study. Subjects attended follow-up visits on a monthly basis until final outcome: healing of the foot, lower-leg amputation, death or non-healing after 1 year. During all visits, data were recorded on standardized case record forms by investigators who were trained during several plenary meetings and on-site visits. Unique aspects of the study were its size, the pan-European scope and the integrated/holistic approach for a multi-organ disease. In this review, the main findings of the study and its implications for diabetic foot care are discussed.